CREST in the Nucleus Accumbens Core Regulates Cocaine Conditioned Place Preference, Cocaine-Seeking Behavior, and Synaptic Plasticity.

Epigenetic mechanisms result in persistent changes at the cellular level that can lead to long-lasting behavioral adaptations. Nucleosome remodeling is a major epigenetic mechanism that has not been well explored with regards to drug-seeking behaviors. Nucleosome remodeling is performed by multi-subunit complexes that interact with DNA or chromatin structure and possess an ATP-dependent enzyme to disrupt nucleosome-DNA contacts and ultimately regulate gene expression. Calcium responsive transactivator (CREST) is a transcriptional activator that interacts with enzymes involved in both histone acetylation and nucleosome remodeling. Here, we examined the effects of knocking down CREST in the nucleus accumbens (NAc) core on drug-seeking behavior and synaptic plasticity in male mice as well as drug-seeking in male rats. Knocking down CREST in the NAc core results in impaired cocaine-induced conditioned place preference (CPP) as well as theta-induced long-term potentiation in the NAc core. Further, similar to the CPP findings, using a self-administration procedure, we found that CREST knockdown in the NAc core of male rats had no effect on instrumental responding for cocaine itself on a first-order schedule, but did significantly attenuate responding on a second-order chain schedule, in which responding has a weaker association with cocaine. Together, these results suggest that CREST in the NAc core is required for cocaine-induced CPP, synaptic plasticity, as well as cocaine-seeking behavior.SIGNIFICANCE STATEMENT This study demonstrates a key role for the role of Calcium responsive transactivator (CREST), a transcriptional activator, in the nucleus accumbens (NAc) core with regard to cocaine-induced conditioned place preference (CPP), self-administration (SA), and synaptic plasticity. CREST is a unique transcriptional regulator that can recruit enzymes from two different major epigenetic mechanisms: histone acetylation and nucleosome remodeling. In this study we also found that the level of potentiation in the NAc core correlated with whether or not animals formed a CPP. Together the results indicate that CREST is a key downstream regulator of cocaine action in the NAc.

Distinct roles for the deacetylase domain of HDAC3 in the hippocampus and medial prefrontal cortex in the formation and extinction of memory.

Histone deacetylases (HDACs) are chromatin modifying enzymes that have been implicated as powerful negative regulators of memory processes. HDAC3has been shown to play a pivotal role in long-term memory for object location as well as the extinction of cocaine-associated memory, but it is unclear whether this function depends on the deacetylase domain of HDAC3. Here, we tested whether the deacetylase domain of HDAC3has a role in object location memory formation as well as the formation and extinction of cocaine-associated memories. Using a deacetylase-dead point mutant of HDAC3, we found that selectively blocking HDAC3 deacetylase activity in the dorsal hippocampus enhanced long-term memory for object location, but had no effect on the formation of cocaine-associated memory. When this same point mutant virus of HDAC3 was infused into the prelimbic cortex, it failed to affect cocaine-associated memory formation. With regards to extinction, impairing the HDAC3 deacetylase domain in the infralimbic cortex had no effect on extinction, but a facilitated extinction effect was observed when the point mutant virus was delivered to the dorsal hippocampus. These results suggest that the deacetylase domain of HDAC3 plays a selective role in specific brain regions underlying long-term memory formation of object location as well as cocaine-associated memory formation and extinction.

Histone serotonylation in dorsal raphe nucleus contributes to stress- and antidepressant-mediated gene expression and behavior.

Mood disorders are an enigmatic class of debilitating illnesses that affect millions of individuals worldwide. While chronic stress clearly increases incidence levels of mood disorders, including major depressive disorder (MDD), stress-mediated disruptions in brain function that precipitate these illnesses remain largely elusive. Serotonin-associated antidepressants (ADs) remain the first line of therapy for many with depressive symptoms, yet low remission rates and delays between treatment and symptomatic alleviation have prompted skepticism regarding direct roles for serotonin in the precipitation and treatment of affective disorders. Our group recently demonstrated that serotonin epigenetically modifies histone proteins (H3K4me3Q5ser) to regulate transcriptional permissiveness in brain. However, this non-canonical phenomenon has not yet been explored following stress and/or AD exposures. Here, we employed a combination of genome-wide and biochemical analyses in dorsal raphe nucleus (DRN) of male and female mice exposed to chronic social defeat stress, as well as in DRN of human MDD patients, to examine the impact of stress exposures/MDD diagnosis on H3K4me3Q5ser dynamics, as well as associations between the mark and depression-related gene expression. We additionally assessed stress-induced/MDD-associated regulation of H3K4me3Q5ser following AD exposures, and employed viral-mediated gene therapy in mice to reduce H3K4me3Q5ser levels in DRN and examine its impact on stress-associated gene expression and behavior. We found that H3K4me3Q5ser plays important roles in stress-mediated transcriptional plasticity. Chronically stressed mice displayed dysregulated H3K4me3Q5ser dynamics in DRN, with both AD- and viral-mediated disruption of these dynamics proving sufficient to attenuate stress-mediated gene expression and behavior. Corresponding patterns of H3K4me3Q5ser regulation were observed in MDD subjects on vs. off ADs at their time of death. These findings thus establish a neurotransmission-independent role for serotonin in stress-/AD-associated transcriptional and behavioral plasticity, observations of which may be of clinical relevance to human MDD and its treatment.

Post-translational modifications of histone proteins by monoamine neurotransmitters.

Protein monoaminylation is a biochemical process through which biogenic monoamines (e.g., serotonin, dopamine, histamine, etc.) are covalently bonded to certain protein substrates via Transglutaminase 2, an enzyme that catalyzes the transamidation of primary amines to the γ-carboxamides of glutamine residues. Since their initial discovery, these unusual post-translational modifications have been implicated in a wide variety of biological processes, ranging from protein coagulation to platelet activation and G-protein signaling. More recently, histone proteins - specifically histone H3 at glutamine 5 (H3Q5) - have been added to the growing list of monoaminyl substrates in vivo, with H3Q5 monoaminylation demonstrated to regulate permissive gene expression in cells. Such phenomena have further been shown to contribute critically to various aspects of (mal)adaptive neuronal plasticity and behavior. In this short review, we examine the evolution of our understanding of protein monoaminylation events, highlighting recent advances in the elucidation of their roles as important chromatin regulators.

Flexible and site-specific manipulation of histones in live animals.

Recent advances in protein engineering have provided a wealth of methods that allow for the site-specific manipulation of proteins in vitro and in cells. However, the efforts to expand these toolkits for use in live animals has been limited. Here, we report a new method for the semi-synthesis of site-specifically modified and chemically defined proteins in live animals. Importantly, we illustrate the usefulness of this methodology in the context of a challenging, chromatin bound N-terminal histone tail within rodent postmitotic neurons located in ventral striatum (Nucleus Accumbens/NAc). This approach provides the field with a precise and broadly applicable methodology for manipulating histones in vivo, thereby serving as a unique template towards examining chromatin phenomena that may mediate transcriptomic and physiological plasticity within mammals.

Histone H3 serotonylation dynamics in dorsal raphe nucleus contribute to stress- and antidepressant-mediated gene expression and behavior.

Background: Major depressive disorder (MDD), along with related mood disorders, is a debilitating illness that affects millions of individuals worldwide. While chronic stress increases incidence levels of mood disorders, stress-mediated disruptions in brain function that precipitate these illnesses remain elusive. Serotonin-associated antidepressants (ADs) remain the first line of therapy for many with depressive symptoms, yet low remission rates and delays between treatment and symptomatic alleviation have prompted skepticism regarding precise roles for serotonin in the precipitation of mood disorders. Our group recently demonstrated that serotonin epigenetically modifies histone proteins (H3K4me3Q5ser) to regulate transcriptional permissiveness in brain. However, this phenomenon has not yet been explored following stress and/or AD exposures. Methods: We employed a combination of genome-wide and biochemical analyses in dorsal raphe nucleus (DRN) of male and female mice exposed to chronic social defeat stress to examine the impact of stress exposures on H3K4me3Q5ser dynamics, as well as associations between the mark and stress-induced gene expression. We additionally assessed stress-induced regulation of H3K4me3Q5ser following AD exposures, and employed viral-mediated gene therapy to reduce H3K4me3Q5ser levels in DRN and examine the impact on stress-associated gene expression and behavior. Results: We found that H3K4me3Q5ser plays important roles in stress-mediated transcriptional plasticity. Chronically stressed mice displayed dysregulated H3K4me3Q5ser dynamics in DRN, with both AD- and viral-mediated disruption of these dynamics proving sufficient to rescue stress-mediated gene expression and behavior. Conclusions: These findings establish a neurotransmission-independent role for serotonin in stress-/AD-associated transcriptional and behavioral plasticity in DRN.

ZBTB7A regulates MDD-specific chromatin signatures and astrocyte-mediated stress vulnerability in orbitofrontal cortex.

Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.

Rescue of deficits by Brwd1 copy number restoration in the Ts65Dn mouse model of Down syndrome.

With an incidence of ~1 in 800 births, Down syndrome (DS) is the most common chromosomal condition linked to intellectual disability worldwide. While the genetic basis of DS has been identified as a triplication of chromosome 21 (HSA21), the genes encoded from HSA21 that directly contribute to cognitive deficits remain incompletely understood. Here, we found that the HSA21-encoded chromatin effector, BRWD1, was upregulated in neurons derived from iPS cells from an individual with Down syndrome and brain of trisomic mice. We showed that selective copy number restoration of Brwd1 in trisomic animals rescued deficits in hippocampal LTP, cognition and gene expression. We demonstrated that Brwd1 tightly binds the BAF chromatin remodeling complex, and that increased Brwd1 expression promotes BAF genomic mistargeting. Importantly, Brwd1 renormalization rescued aberrant BAF localization, along with associated changes in chromatin accessibility and gene expression. These findings establish BRWD1 as a key epigenomic mediator of normal neurodevelopment and an important contributor to DS-related phenotypes.