2006 — 2010 |
West, Anne Elizabeth |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Epigenetic Regulation of Transcriptional Repression by Drugs of Abuse
Alterations in gene expression contribute to the adaptations of brain reward circuits that underlie persistent behaviors associated with drug abuse. Epigenetic regulation of transcription mediated by posttranslational modification of histone proteins is emerging as an important mechanism used by environmental factors, including drugs of abuse, to control gene expression. Identifying the histone modifying factors that respond to drugs of abuse and understanding how chromatin regulation affects behavior are crucial for making progress toward understanding the neural basis of drug addiction. One intriguing candidate for this process is the methyl-DNA binding transcriptional represser MeCP2 as this protein plays important roles in brain development and function. MeCP2 is known to repress its target genes by recruiting both histone deacetylase and methyltransferase enzymes. Interestingly, given the conservation between activity-dependent and drug-induced mechanisms of transcriptional regulation, we have recently found that MeCP2 is phosphorylated in an activity-dependent manner in neurons at a site that dynamically modulates the ability of MeCP2 to repress its target gene Bdnf. We hypothesize that MeCP2 is a key mediator of cocaine-regulated transcription, and that the regulation of repressive histone methylation contributes to the development of drug sensitization behaviors. We propose to test this hypothesis by combining biochemical analyses of cocaine-dependent regulation of MeCP2 and histone methylation with experimental manipulation of these processes in mouse models of drug abuse. Our specific aims are: 1) to examine the functional regulation of MeCP2 by drugs of abuse;2) to investigate the contribution of epigenetic transcriptional repression mechanisms to cocaine-regulated gene expression;and 3) to evaluate the contribution of epigenetic mechanisms of gene transcription to drug-induced behavioral sensitization. These studies will fill a critical gap in knowledge by defining new mechanisms that contribute to the effects of cocaine on both neuronal gene expression and behavior. The identification of epigenetic mechanisms involved in regulation of gene expression by drugs of abuse may reveal new targets for therapies in treatment of drug addiction.
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2012 — 2013 |
West, Anne Elizabeth |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Regulation of Response to Chronic Antidepressant Treatment by Mecp2
DESCRIPTION (provided by applicant): Most currently available antidepressant drugs rapidly alter neurotransmission in the brain yet require chronic administration to affect mood. These observations suggest that slow biological processes downstream of drug- induced changes in neurotransmission, which are thought to include changes in gene transcription, are likely to be required for the behavioral response to these antidepressant drugs. The goal of this proposal is to advance understanding of the basic brain mechanisms that regulate behavioral responses to chronic antidepressant treatment. We have discovered that the methyl-DNA binding chromatin regulatory protein MeCP2 is a target of regulation by antidepressants, and that mutations in MeCP2 alter behavioral responses to chronic antidepressant treatment in mice. Specifically we find that administration of drugs that enhance serotonin and dopamine neurotransmission in the CNS, including the antidepressant citalopram, induces phosphorylation of MeCP2 at Ser421 (pMeCP2) in selected CNS neurons. To test the functional relevance of pMeCP2 for depressive-like behaviors and antidepressant responses we obtained a strain of mice that bear a germline Ser421Ala mutation knocked into the Mecp2 gene that renders MeCP2 protein nonphosphorylatable at this site. We have tested the consequence of this mutation on depressive-like behaviors and response to chronic antidepressant treatment in these mice in the social defeat stress paradigm. We find that both MeCP2 knockin mice and their wildtype littermates show similar levels of social avoidance following defeat. However whereas chronic imipramine treatment rescues social interaction in the wildtype mice, this drug treatment fails to ameliorate social avoidance in the knockin mice. On the basis of these and other preliminary data we hypothesize that phosphorylation of MeCP2 at Ser421 is required for at least a subset of behavioral responses to chronic antidepressant treatment. This is a completely novel hypothesis. No prior studies have examined a role for MeCP2 in depressive-like behaviors or response to antidepressant treatment. Our findings could have a substantial impact on the understanding of brain processes that underlie depressive-like behaviors and response to antidepressants by linking this important epigenetic regulator of chromatin to the actions of chronic antidepressant administration on the brain. We propose to address our hypothesis with the following two specific aims: 1) To analyze depressive-like behaviors and antidepressant responses in MeCP2 KI mice and 2) To identify neural circuits that underlie MeCP2-dependent effects on depressive-like behaviors.
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2012 — 2013 |
West, Anne Elizabeth |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Amphetamine-Induced Transcriptional Plasticity in Striatal Gabaergic Interneurons
The mesolimbic reward circuit is comprised of dopaminergic neurons in the ventral tegmental area and their targets in the nucleus accumbens (NAc) and other associated limbic brain regions. This circuit is the major site of action for addictive drugs such as psychostimulant amphetamine (AMPH). The reinforcing properties of AMPH are mediated by changes in the physiology and synaptic conectivity of neurons in the NAc. Considerable evidence suggests that AMPH-induced changes in striatal gene expression are essential for these cellular adaptations, and chromatin regulation has been implicated as a mechanism that may contribute to the persistence of these changes in neuronal physiology. However the striatum is comprised of multiple kinds of neurons that are synaptically interconnected into functional microcircuits, and very little is known about whether or how cell-type specific differences in AMPH-regulated transcription impact striatal function. We propose to take a novel approach to this question by using a protocol we have developed for fluorescence- activated cell sorting (FACS) to characterize AMPH-induced changes in gene transcription in striatal fast- spiking GABAergic interneurons (FSIs). FSIs play a crucial role in gating striatal output, however it remains unknown whether these neurons experience AMPH-dependent adaptations. The diffuse distribution of FSIs has presented a significant barrier to biochemical analysis of their gene transcription and chromatin regulation. However we discovered that AMPH administration drives rapid and robust phosphorylation of the methyl-DNA binding protein MeCP2 at Ser421 (pMeCP2) in the NAc, and that this AMPH-induced phosphorylation occurs selectively in FSIs. On the basis of these findings we have developed FACS protocols to use the pMeCP2 antibody as a label to purify AMPH-activated FSIs from the mouse striatum. Here we propose to use this technique in order to determine whether FSIs show plasticity of transcriptional regulation in response to repeated AMPH exposure. In Aim 1 we will FACS purify FSIs from the striatum of mice that received either acute or repeated AMPH injections and profile changes in gene expression by RNA-Seq. In Aim 2 we will FACS purify FSI nuclei from the striatum of mice that received either acute or repeated AMPH injections and perform ChIP-Seq with antibodies against acetylated histone H3 as an indication of chromatin regulation. If we observe differences in gene expression or chromatin regulation, these data would provide the first evidence that this interneuron population experiences molecular adaptations in response to repeated AMPH exposure. This outcome would be exciting because it would raise the posibility that transcriptional plasticity is accompanied by AMPH-induced adaptations in FSI function. We anticipate that identifying FSI genes regulated by repeated AMPH will suggest new hypotheses of how plasticity of this important interneuron population may contribute to AMPH-induced changes in mesolimbic circuit functions.
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2013 — 2014 |
Crawford, Gregory E (co-PI) [⬀] West, Anne Elizabeth |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Genome-Wide Mapping of Enhancer Elements For Neuronal Differentiation Genes
DESCRIPTION (provided by applicant): Cellular differentiation requires the precise orchestration of gene expression programs. Chromatin regulatory complexes coordinate this process by modulating the accessibility and activation state of gene regulatory elements. The end states of cellular differentiation can be readily visualized through the comparison of cell typ specific epigenome maps. Such analyses indicate that the differential regulation of distal enhancer elements is the primary determinant of cell-type specific programs of gene expression. However the dynamic chromatin regulatory events that sculpt the distribution of active enhancers and thus drive the differentiation of any single cell type have remained largely unknown. The goal of this proposal is to identify the chromatin regulatory events that control the differentiation of neurons in vivo. We will identify developmentally regulated chromatin changes that control contemporaneous changes in neuronal gene expression by comparing epigenomic profiles of chromatin harvested from discrete stages in the differentiation of a specific neuronal cell type. Cerebellar granule neurons (CGNs) provide an ideal in vivo model for this study because they represent a largely homogeneous neuronal population that can be obtained in very large numbers at discrete developmental stages directly from the postnatal mouse brain. Using the technique of DNaseI chromatin digestion followed by high-throughput sequencing (DNase-Seq) we have already identified substantial differences in the distribution of accessible chromatin over the course of CGN differentiation. Here we propose to characterize the developmental regulation of promoters and enhancers in these neurons by performing genome-wide chromatin immunoprecipitation (ChIP) for histone marks that denote the nature and activation state of these gene regulatory elements. We will then test the relationship between chromatin states and dynamic changes in gene expression during CGN differentiation through hidden Markov modeling of our combined DNase-Seq, ChIP-Seq, and RNA-Seq datasets. The outcome of this proposal will be the first identification of a comprehensive defined set of gene regulatory elements that control the dynamic changes in gene regulation that underlie neuronal differentiation.
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2013 — 2017 |
West, Anne Elizabeth |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Cocaine Reward and Reinforcement by Mecp2
DESCRIPTION (provided by applicant): Chronic cocaine abuse arises as a result of persistent cocaine-induced adaptations in the function of neurons within mesolimbocortical brain reward circuits. An understanding of the molecular mechanisms by which cocaine alters the function of these neural circuits may lead to development of novel therapies for the treatment of cocaine addiction. The overall hypothesis of this proposal is that cocaine exerts long-lasting effects on behavior by inducing the transcription of new gene products in the nucleus accumbens (NAc) that change the excitability and/or synaptic connectivity of NAc neurons. We have shown that genetically manipulating the expression of the methyl-DNA binding protein MeCP2 in specific regions of the adult striatum modulates the ability of cocaine and the related psychostimulant amphetamine to induce addictive-like behaviors in rodents. Furthermore, we find that cocaine induces phosphorylation of MeCP2 at Ser421 (pMeCP2) selectively in parvalbumin (Pv)-positive fast-spiking GABAergic interneurons (FSIs) in the NAc. The objective of this proposal is to test the novel hypothesis that phosphorylation of MeCP2 in FSIs provides a mechanism to link cocaine exposure with transcription-dependent changes in striatal circuit function that limit the rewarding properties of cocaine. To address this hypothesis, in Aim 1 we will test the consequences of MeCP2 phosphorylation on the rewarding and reinforcing properties of cocaine by assessing cocaine self-administration (SA) in mice bearing a mutation knocked into the Mecp2 gene that changes Ser421 to Ala, rendering MeCP2 non-phosphorylatable at this site. In Aim 2 we will test the hypothesis that MeCP2 acts in FSIs in the NAc to limit cocaine SA. We will achieve this goal by stereotaxically injecting LoxP-conditional viruses into the NAc of adult mice from a transgenic strain that expresses the Cre recombinase in Pv-positive neurons. We will use these viruses to manipulate MeCP2 expression and phosphorylation in these cells as a means to alter FSI function and we will determine the effects of these manipulations on cocaine SA. Finally in Aim 3 we will test the hypothesis that pMeCP2 regulates a striatal plasticity that limits cocaine SA by modulating the inducibility of immediate-early genes in FSIs of the NAc. The outcome of our study will be the experimental demonstration of a specific circuit-based mechanism by which the chromatin regulatory protein MeCP2 limits cocaine reward. These studies promise to yield significant new insights into the neurobiological mechanisms that impact susceptibility to cocaine addiction.
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2016 — 2017 |
Gersbach, Charles A (co-PI) [⬀] West, Anne Elizabeth |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
In Vivo Epigenome Editing With Crispr-Based Histone Acetyltransferase Transgenic Mice
? DESCRIPTION: Chronic cocaine abuse arises as a result of persistent cocaine-induced adaptations in the function of the neurons that comprise mesolimbocortical brain reward circuits. Cocaine-induced changes in gene transcription contribute to many of these alterations in neuronal function. Furthermore cocaine exposure has been shown to dynamically alter the epigenome by regulating the expression and/or function of histone and DNA modifying enzymes. Taken together, these data have led to the hypothesis that long-lasting changes in the epigenome may underlie the persistence of cocaine-induced addictive-like behaviors. However whether specific changes in chromatin regulation are truly causative for drug-induced behavioral plasticity has remained a challenging hypothesis to test due to the lack of high-throughput in vivo methods for site-specific experimental manipulation of the epigenome. To overcome this limitation we will generate two novel Cre/loxP-conditional CRISPR/Cas9- based transgenic mouse strains in which an enzymatically dead Cas9 protein fused either to the core histone acetyltransferase domain of p300 (dCas-p300) or the KRAB repressor domain (dCas9-KRAB) is knocked into the Rosa26 locus. We have shown that gRNA-mediated recruitment of dCas9 fusions with chromatin regulators is sufficient to induce targeted histone modifications and highly specific corresponding changes in gene transcription. Now by expressing these fusion proteins in transgenic mice, we will achieve regional and temporal control of site-specific epigenome editing in the brain in vivo by intersecting Cre-dependent induction of dCas9-fusion protein expression with stereotaxic viral delivery of validated gRNAs targeting cocaine- regulated enhancers. In the R21 phase of this proposal we will first generate and characterize the conditional dCas9-p300 and dCas9-KRAB mouse strains and then conduct a proof-of-principle experiment to validate whether dCas9-mediated regulation of Fosb in neurons of the nucleus accumbens is sufficient to alter cocaine- induced locomotor sensitization and conditioned place preference. In the R33 phase we will use the dCas9- p300 and dCas9-KRAB mouse strains to test the hypothesis that epigenetic sensitization of Bdnf transcription in dopaminergic neurons of the ventral tegmental area promotes incubation of cocaine craving, an important rodent model for relapse. Finally to accelerate future epigenome editing studies we will first use a novel method to capture nuclei of cocaine-activated neuronal ensembles in the nucleus accumbens for chromatin profiling and then develop and functionally validate gRNAs targeting cocaine-regulated enhancers for use by the broader scientific community. Our studies will provide a novel toolbox for functional epigenomic studies of the molecular mechanisms underlying substance use disorders.
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2017 — 2021 |
West, Anne Elizabeth |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Chromatin Mechanisms of Neuronal Maturation
Neuronal differentiation requires the precise orchestration of gene expression programs. Temporal control of transcription is particularly important in maturing postmitotic neurons of the postnatal brain because these cells need to express gene products that refine neuronal function and circuitry during specific critical periods of brain development. Epigenetic regulation of chromatin structure is known to play a role in establishing cell-type specific programs of gene expression during early stages of cell fate determination, but the chromatin mechanisms that regulate gene expression during terminal phases of differentiation remain to be fully understood. The goal of this proposal is to identify chromatin mechanisms that coordinate the induction of genes in maturing neurons of the CNS. To discover these chromatin mechanisms of neuronal differentiation, and to determine how these mechanisms are regulated across multiple stages of neuronal maturation, we have characterized chromatin accessibility, enhancer activation, and gene expression in differentiating cerebellar granule neurons (CGNs) of the postnatal mouse in vivo. We have observed that thousands of regulatory elements show chromatin accessibility changes as CGNs differentiate, and we have verified that many of these differentially accessible regions function as developmental stage-specific enhancers of neuronal genes. Furthermore our data identify a specific group of the genes selectively expressed in mature CGNs of the cerebellum that are associated with repressive histone H3 lysine 27 trimethylation (H3K27me3) at early postmitotic stages of CGN differentiation yet lose this mark as CGNs mature. Consistent with a role for active histone demethylation in the induction of these genes, we find that at least a subset of these genes show increased H3K27me3 and reduced mRNA expression in CGNs lacking the H3K27 demethylase Kdm6b. The overarching hypothesis of this proposal is that the molecular regulators of H3K27me3 dynamics in postmitotic neurons play a key role in timing neuronal maturation. To address this hypothesis we propose the following Specific Aims: 1) To characterize the dynamic demethylation of H3K27 in postmitotic neurons maturing in vivo, 2) To determine the role of the H3K27 demethylase Kdm6b in neuronal maturation, and 3) To identify enhancer mechanisms of transcriptional derepression in maturing neurons. Together these studies will identify the molecular mechanisms that mediate the gene-specific loss of H3K27me3 in maturing CGNs, and they will directly test the importance of the developmental derepression of H3K27me3-silenced genes for functional neuronal maturation.
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2018 — 2020 |
Gersbach, Charles A. (co-PI) [⬀] West, Anne Elizabeth |
R33Activity Code Description: The R33 award is to provide a second phase for the support for innovative exploratory and development research activities initiated under the R21 mechanism. Although only R21 awardees are generally eligible to apply for R33 support, specific program initiatives may establish eligibility criteria under which applications could be accepted from applicants demonstrating progress equivalent to that expected under R33. |
In Vivo Epigenome Editing With Crispr-Based Histone Acetyltransferase Transgenic
? DESCRIPTION: Chronic cocaine abuse arises as a result of persistent cocaine-induced adaptations in the function of the neurons that comprise mesolimbocortical brain reward circuits. Cocaine-induced changes in gene transcription contribute to many of these alterations in neuronal function. Furthermore cocaine exposure has been shown to dynamically alter the epigenome by regulating the expression and/or function of histone and DNA modifying enzymes. Taken together, these data have led to the hypothesis that long-lasting changes in the epigenome may underlie the persistence of cocaine-induced addictive-like behaviors. However whether specific changes in chromatin regulation are truly causative for drug-induced behavioral plasticity has remained a challenging hypothesis to test due to the lack of high-throughput in vivo methods for site-specific experimental manipulation of the epigenome. To overcome this limitation we will generate two novel Cre/loxP-conditional CRISPR/Cas9- based transgenic mouse strains in which an enzymatically dead Cas9 protein fused either to the core histone acetyltransferase domain of p300 (dCas-p300) or the KRAB repressor domain (dCas9-KRAB) is knocked into the Rosa26 locus. We have shown that gRNA-mediated recruitment of dCas9 fusions with chromatin regulators is sufficient to induce targeted histone modifications and highly specific corresponding changes in gene transcription. Now by expressing these fusion proteins in transgenic mice, we will achieve regional and temporal control of site-specific epigenome editing in the brain in vivo by intersecting Cre-dependent induction of dCas9-fusion protein expression with stereotaxic viral delivery of validated gRNAs targeting cocaine- regulated enhancers. In the R21 phase of this proposal we will first generate and characterize the conditional dCas9-p300 and dCas9-KRAB mouse strains and then conduct a proof-of-principle experiment to validate whether dCas9-mediated regulation of Fosb in neurons of the nucleus accumbens is sufficient to alter cocaine- induced locomotor sensitization and conditioned place preference. In the R33 phase we will use the dCas9- p300 and dCas9-KRAB mouse strains to test the hypothesis that epigenetic sensitization of Bdnf transcription in dopaminergic neurons of the ventral tegmental area promotes incubation of cocaine craving, an important rodent model for relapse. Finally to accelerate future epigenome editing studies we will first use a novel method to capture nuclei of cocaine-activated neuronal ensembles in the nucleus accumbens for chromatin profiling and then develop and functionally validate gRNAs targeting cocaine-regulated enhancers for use by the broader scientific community. Our studies will provide a novel toolbox for functional epigenomic studies of the molecular mechanisms underlying substance use disorders.
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2019 — 2021 |
West, Anne Elizabeth |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Psychostimulant-Induced Plasticity of Nucleus Accumbens Interneurons
Psychostimulant drugs of abuse induce persistent changes in the function of neural reward circuits that underlie the development of addiction. The nucleus accumbens (NAc) plays a significant role in motivation, reward, and reinforcement learning, and this brain region is a major site of the psychostimulant-induced cellular adaptations that lead to drug addiction. Substantial data indicate that changes in gene transcription, mediated by psychostimulant-dependent regulation of chromatin, play a key role in driving persistent changes in NAc function. Though many past studies have focused on the induction of these transcriptional and chromatin regulatory events in spiny projections neurons (SPNs) of the NAc, the NAc is comprised of multiple cell types, and the output of the NAc is powerfully modulated by the activity of several classes of interneurons. We have shown that silencing the function of one of these interneuron populations, the parvalbumin (PV)-expressing population of NAc GABAergic interneurons, blocks the expression of locomotor sensitization and conditioned place preference (CPP) induced by repeated amphetamine exposure in mice. Functional plasticity of striatal PV+ interneurons has also been implicated in both cocaine self-administration and habit learning, suggesting a conserved function for these neurons in the circuit adaptations underlying a number of motivational behaviors. Nonetheless, we know very little about the molecular mechanisms by which psychostimulants modulate the functional plasticity of PV+ interneurons to effect changes in addictive-like behaviors. In order to identify and link PV+ interneuron molecular plasticities to the cellular and circuit adaptations in NAc that underlie addictive- like behaviors, we must identify cell type specific programs of chromatin regulation and gene transcription and determine their functional consequences. Here we will create this roadmap from transcription through molecular mediators to behavior. We will use PV+-interneuron specific identification and manipulations of AMPH-regulated genes in vivo and study convergent effects on the physiology of NAc PV+ interneurons and the sensitivity of mice to AMPH-induced CPP. The goal of this proposal is to test the overarching hypothesis that psychostimulant-dependent regulation of transcription in NAc PV+ interneurons alters the function of these neurons to slow the development of addictive-like behaviors. In Aim 1 we will conduct a specific test of this hypothesis by determining the functional importance of the perineuronal net protein Brevican as a psychostimulant-regulated modulator of PV+ interneuron synaptic plasticity and addictive-like behaviors. In Aim 2 we will use leading edge epigenome-editing and chromatin analysis methods to discover more broadly how psychostimulant-dependent transcription factor induction in PV+ interneurons of the NAc coordinates downstream programs of gene expression to mediate long-lasting changes in PV+ neuron function. Taken together these studies will reveal how cellular plasticity mechanisms act within PV+ interneurons of the NAc to gate the adaptations of NAc function that underlie addictive-like behaviors.
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