Evan Deneris - US grants

Molecular cloning studies have identified gene families encoding functionally distinct heteromeric ligand-gated ion-channels. One such family of genes encodes subunits that are assembled into several functionally-distinct heteromeric neuronal nicotinic acetylcholine receptors. The nicotinic receptor subunit gene family is abundantly expressed in the vertebrate brain and peripheral nervous system. Each member has a unique pattern of expression, although these patterns overlap partially. Thus, this gene family is likely to encode multiple heteromeric receptors to subserve synaptic functions in numerous cholinergic pathways of the brain and periphery. This proposal is concerned with identifying the mechanisms that control vertebrate receptor subunit gene expression so that functionally distinct heteromeric nicotinic receptors can be assembled in different subsets of phenotypically distinct neurons. The experiments described are designed to: 1) identify the cis-acting elements that control neuronal nicotinic receptor subunit gene transcription in a neural-specific manner, 2) determine whether the influence of NGF on nicotinic receptor subunit RNAs is exerted at the level of transcription or RNA stability, 3) determine the influence of activity on receptor subunit gene expression, and 4) determine if changes in receptor subunit gene expression occur during periods of rapid synaptogenesis. The gene family encoding neuronal nicotinic receptor subunits are particularly amenable to the proposed investigation because its members are expressed in a wellcharacterized clonal cell line, PC12 and in the accessible and experimentally manipulatable sympathetic nervous system. The PC12 line provides a convenient cell culture system in which to begin characterizing transcription regulatory elements controlling ligand-gated ion-channel genes. Sympathetic ganglia provide an accessible experimental system of primary neurons for complementary in vivo and in vitro approaches to address the influence of cell-cell interactions on receptor subunit gene expression. Neuronal nicotinic receptor subunit genes and other ligand-gated ion-channel genes are co-expressed in numerous nuclei and cell layers of the brain. Common neuronal environments raise the possibility that the genes encoding different ligand-gated ion-channels share similar mechanisms of regulation. Thus, this investigation is likely to serve as a model for the regulation of other ligand-gated ion-channel genes, for which well-characterized and accessible experimental models systems are not as well developed. The investigation of nicotinic receptor gene expression will ultimately lead to an understanding of the importance of receptor gene regulatory mechanisms to the development of the nervous system and perhaps insight into the molecular mechanisms of neurological disorders which result from aberrant development.

Our general long-term objective is to understand the transcriptional mechanisms that control cell-type diversity within the nervous system. To pursue this objective we are investigating cis and trans transcriptional control of genes encoding neuronal nicotinic receptors. These genes encode subunits that can be assembled into a variety of functionally distinct heteromeric excitatory ligand-gated ion channels. Expression patterns of these genes indicates that different heteromers are expressed in adrenal chromaffin cells, peripheral ganglia, retina, and throughout the brain. The focus of our research is a cluster of nAchR genes, beta4, alpha3, and alpha5 that encode subunits assembled into a single receptor subtype in ganglia and possibly central neurons. The basic question driving our research is how are members of this cluster coordinately controlled to generate requisite overlapping patterns of expression for heteromer assembly? Coexpression and the clustered organization suggest that these genes are subject to control by shared cis elements. However, expression patterns of these genes are not entirely concordant and therefore individual genes in the cluster are likely to be controlled by independent cis elements as well. We have identified independent promoters adjacent to the alpha3 and beta4 genes as well as a potential enhancer within the beta4/alpha3 intergenic region. Our interest now is to investigate these cis elements in nAchR expressing PC12 cells to define their functional properties and in transgenic animal to determine their role in neural- specific expression of these nAchR genes. We have also identified trans- acting factors that modulate alpha3 and beta4 promoter activity. The zinc- finger protein Sp1 or an Sp1-related factor transactivates the alpha3 promoter via a G+A-rich motif positioned adjacent to the alpha3 transcription start site region. Sp1 belongs to a differentially expressed gene family and therefore one goal is to identify Sp1 family members that are expressed in PC12 cells and to assess their function in nAchR transcription. Toward this coal we have found that the Sp1-related factor, Sp4, is coexpressed with Sp1 in PC12 cells. Thus we will investigate the expression and function of this second zinc-finger in nAchR gene transcription. We will also extend these studies to the beta4 promoter in order to determine whether alpha3 and beta4 are coordinately controlled by these proteins. We have discovered that a POU-domain transcription factor, SCIP/Tst-1/Oct-6, is a potent and specific activator of alpha3. This represents the first cellular gene identified that is positively modulated by SCIP and it raises the possibility that SCIP controls cholinoceptive phenotype in neurons. Our recent studies in PC12 cells indicate that alpha3 regulation by SCIP is cell-type specific and suggest that activation occurs via a novel mechanism. We are interested in using PC12 cells as a neural model to investigate the potential alternative mechanism of SCIP action on alpha3 and other promoters. Together the proposed work will lead to a better understanding of how cholinergic transmitter systems are built and more generally will help to provide a clear view of the control of gene expression in neurons. Ultimately, these studies are likely to establish a foundation for future investigations of the role of aberrant gene control in specific neurological disorders.

DESCRIPTION (Adapted from applicant's abstract): Pet-1 is a new ETS domain transcription factor and is a rare example of a factor that appears to be expressed in a single brain neurotransmitter system. Pet-1 is expressed in the brain virtually exclusively in the 5-HT system. Preliminary analysis indicates that Pet-1 is expressed in most or all raphe nuclei, including the laterally extending B9 cluster. The sites of Pet-1 expression in the reticular formation map very well to the 5-HT neuron clusters classified as the B3 group. Thus the pattern of Pet-1 expression in the brain is limited to most and perhaps all 5-HT nuclei in the brainstem. Based on the likely role of Pet-1 as a sequence-specific transcriptional activator and its provocative expression pattern, it is hypothesized that Pet-1 functions to control either the specification, proliferation, or differentiation of serotonergic neurons in the vertebrate CNS. The detection of Pet-1 RNA in E14 rat head is consistent with a function early in development. Pet-1 may be a precise marker for all or part of the 5-HT neuron lineage. It may mark the earliest serotonergic neuron progenitors or cells at a later stage in the differentiation of the lineage. The possible developmental role of Pet-1 will be investigated by determining when and where it is expressed in the fetal brain. In situ hybridization and immunohistochemistry will be used to precisely determine the expression of Pet-1 in the 5-HT system. The onset of Pet-1 expression will be determined and compared to the appearance of 5-HT in the brainstem. Pet-1-specific polyclonal antisera will be generated to provide an immunological reagent for the detection of Pet-1 in the brainstem and for biochemical analysis of its function. The proposed experiments will suggest likely functional roles of Pet-1 in the developing brainstem and will facilitate interpretation of mutant phenotype in future gene targeting of the murine Pet-1 locus. The identification and characterization of a transcription factor that is likely to be a key regulator of the vertebrate CNS 5-HT system has clear significance for elucidating the molecular mechanisms governing the development of this vital neurotransmitter system. This system is involved in the control of numerous neural functions such as cognition, affect, pain, appetite, sex, sleep, aggression, and perception. Abnormal function of the 5-HT system has been implicated in numerous psychiatric disorders. Future loss or gain of function approaches to the study of Pet-1 in the mouse may create novel models for clinical disorders involving the 5-HT system.

DESCRIPTION (From applicant's abstract): This application proposes a unique avenue of research aimed at the development and function of the mammalian serotonin neurotransmitter system. The research is based on our discovery of a novel ETS domain transcription factor, Pet-1, whose expression precisely marks 5-HT neurons before 5-HT appears in the hindbrain and before 5-HT neurons have begun to migrate to their adult midbrain/hindbrain positions. The discovery of a transcription fact that is specifically expressed in the mammalian 5-HT system is unprecedented. It serotonergic-specific expression pattern together with the presence of a Pet-1 DNA binding sites in human and mouse genes, which in large part defines the differentiated phenotype of 5-HI neurons, suggest that Pet-1 is a key determinant in the decision to become a 5-HT neuron. The experiments described in this application are aimed at testing this hypothesis and to exploit the provocative expression pattern of Pet-1 to control heterologous gene expression in a serotonergic-specific manner. The proposed aims are: 1) Investigate expression of Pet-1 alternative forms and expression of other ETS factors in the 5-HT system. 2) Identify a Pet genomic DNA fragment capable of controlling serotonergic-specific gene expression. 3) Investigate the transcriptional interaction of Pet-1 with the serotonin transporter (5-HTT), 5 HT1a receptor and tryptophan hydroxlase (TPH) genes. 4) Create a Pet-1 loss of function mouse and determine its phenotype. The function of the central 5-HT system is abnormal in numerous psychiatric disorders and thus this system is the target of several highly effective pharmacological agents that are used to treat these disorders. The information gained from completion of the proposed aims is likely to have significant impact on our basic understanding of the 5-HT system and may lead in the future to the creation of novel animal models for psychiatric disorders involving the 5-HT system.

DESCRIPTION (provided by applicant): The objective of the research proposed here is to understand transcriptional mechanisms that control cholinergic neurotransmitter system development in vertebrates. A conserved cluster of vertebrate genes ordered beta4, alpha3, and alpha5 encode subunits that are assembled into several neuronal nicotinic acetylcholine receptor (nAchR) subtypes in peripheral and central neurons. One subtype composed of all three subunits is essential for excitatory post-synaptic transmission in adrenal chromaffin cells and between pre- and post-ganglionic neurons of the sympathetic and parasympathetic nervous systems. Subtypes containing beta4 and alpha3 subunits are important for cholinergic synaptic transmission in retina. Before these subtypes can be assembled the genes encoding beta4, alpha3, and alpha5 must be transcribed in appropriate neurons at the correct time. However, the transcriptional mechanisms that control subunit expression are poorly understood. We have identified an enhancer (beta43') within the cluster that is likely to be important for transcription of the subunit genes in neurons. We also showed that ETS domain factor interactions are important for neuronal activity of beta43'. In this proposal we wish to determine the biological relevance of beta43' and its interactions with ETS factors for the assembly of different receptor subtypes made from beta4, alpha3, and alpha5. Our hypothesis is that beta43' controls neuron specific transcription of the clustered genes through ETS interactions. We have prepared transgenic mice expressing each of the clustered genes from a Pl-bacterial artificial chromosome (PAC). We will prepare additional lines of transgenic mice that carry mutations in the enhancer ETS binding sites. Then transcription of each clustered gene will be investigated in various neuronal cell types that express the cluster. We will also investigate whether the cluster is a transcriptional target of the Pet-1 ETS factor by investigating subunit RNA and protein levels in our Pet-1 knock out mice. We will also investigate ETS factor expression in neuronal cell types that express the cluster. This research will further our understanding of the mechanisms governing the development of neuronal cholinergic systems and assembly of specific nAchR subtypes. It will also help to reveal the poorly defined functions of ETS factors in vertebrate neurons.

DESCRIPTION (provided by applicant): This application proposes to continue research into the transcriptional mechanisms that control development of serotonin (5ht) neuron transmitter traits and 5ht-modulated behaviors. Altered 5ht signaling has been implicated in the pathogenesis of numerous neurological and psychiatric disorders such as autism, sudden infant death syndrome (SIDS), depression, and anxiety. Many of these disorders are neurodevelopmental in origin and risk for acquiring them is influenced by heritable susceptibility factors. Thus, the general concept that stimulates the research proposed here is that genetically driven variation in transcriptional programs governing 5ht neuron development contributes to the pathogenesis of certain neurological and psychiatric disorders by adversely altering 5ht system activity. The focus of the proposed research is the Pet-1 ETS factor whose expression is initiated specifically in postmitotic 5ht neuron precursors before the appearance of 5ht and then maintained in all adult 5ht neurons. Loss of functions studies in the mouse demonstrated that Pet-1 is a critical transcriptional determinant of 5ht neuron phenotype and 5ht-modulated behaviors. These findings support the existence of a Pet-1 dependent transcriptional program that impacts 5ht- modulated behaviors through its control of embryonic 5ht neuron development. The new aims proposed here will address the following questions: 1) Is the FEV ETS factor the functional human orthologue of Pet-1 and does a FEV-dependent genetic program govern human 5ht neuron development? 2) Does the level of Pet- 1/FEV activity determine the level of 5ht neuron transmitter traits and 5ht neuron function? 3) What role does Pet-1 play in mature 5ht neurons? 4) Is the transcriptional control of Pet-1 and FEV conserved? The proposed aims will be investigated with molecular genetic approaches and will be greatly facilitated by our newly developed genetic based tools designed to access 5ht neurons, in vivo. If FEV functions in human 5ht neuron development then alterations in FEV activity brought about either by genetic or environmental factors are a potential source of risk for psychiatric or neurological disease and interindividual variation in behavior. The proposed studies are likely to have a long-term impact on molecular psychiatry research by advancing an understanding of the genetic mechanisms that govern 5ht neuron development and determining how these mechanisms impact behavior and physiology.

genetic regulation; laboratory mouse

DESCRIPTION (provided by applicant): This project is aimed at investigating the genetic mechanisms that act across lifespan to regulate 5-HT system function and determine how these mechanisms impact postnatal 5-HT modulated behaviors. The basis of these studies is the discovery of a Pet-1 dependent transcriptional program that regulates 5-HT-modulated behaviors and newly developed 5-HT neuron-specific and temporally controlled conditional targeting approaches. Our approaches have enabled an investigation of 5-HT neuron-specific transcriptional mechanisms in purified 5-HT neurons and functional studies of serotonergic genes at any stage of life with reliable spatial and temporal control of gene ablation. Many questions remain unanswered about the mechanisms through which Pet-1 controls 5-HT neuron development and ultimately 5-HT modulated behaviors. For example, although our previous studies have shown that Pet-1 is needed for the coordinate induction of genes (Tph2, AADC, Sert, Vmat2, Htr1A, Htr1B, MaoB) directly responsible for 5-HT synthesis, reuptake, autoinhibition and metabolism it is not known whether Pet-1's function is restricted to these genes. It is also not known whether Pet-1 regulates different sets of genes in different populations of 5-HT neurons and whether it is solely an activator of serotonergic gene expression or whether it, perhaps, also simultaneously represses expression of some genes during the development of 5-HT neurons. Although we have shown that Pet-1 is needed in adult 5-HT neurons for maintenance of Tph2 and Sert the critical periods for Pet-1-directed transcription in essential 5-HT neuron functions such as 5-HT neuron firing and 5-HT modulated behaviors are poorly understood. We will investigate whether transcriptional regulation by Pet-1 is required for control of 5-HT signaling at different stages of life including the neonatal to adolescent period. The neonatal period (P5-P20) is a period in which 5-HT signaling has been shown to be critical for normal adult emotional behaviors while the adolescent period coincides with the onset of 5-HT related disorders such as schizophrenia and stress-related emotional disturbances. Finally, the importance of 5-HT synthesis in neonatal and adult 5-HT neurons for 5-HT modulated behaviors has never been adequately addressed because of the lack of suitable methods. We will use our new temporal conditional targeting approaches to test specific hypotheses about the early postnatal and adulthood requirements for 5-HT synthesis in the behaving mouse. Together, our new aims will investigate the downstream network of Pet-1 regulated genes, the requirements for intrinsic transcriptional mechanisms, and 5-HT synthesis at malleable developmental transition periods that are known to be critical periods for the determination of mental health relevant behaviors.

Early life 5-HT signaling influences neurodevelopmental trajectories and altered 5-HT signaling has been implicated in the pathogenesis of numerous stress-related psychiatric disorders. What is not clear is how extracellular 5-HT exerts its effects on nervous system development and whether the critical synthetic source of 5-HT is the brain. In Project 1: Early Brain Serotonin and its Lasting Impact on Neuronal Epigenetic Programming, Evan Deneris seeks to determine whether 5-HT synthesized specifically in hindbrain raphe neurons and secreted during fetal and early postnatal life is an important extracellular signal required for early-life epigenetic programming of serotonergic homeostasis and hypothalamic-pituitary-adrenal (HPA) axis stress circuitry. To investigate this hypothesis, Deneris seeks to apply his recently developed temporally controlled targeting approaches to knock out the gene, tryptophan hydroxylase 2, responsible for synthesis of brain 5-HT. Tph2 will be targeted during fetal and early postnatal life to reduce brain 5-HT synthesis but not synthesis of 5-HT from exogenous sources such as the placental or gut. The targeting of Tph2 at different stages of early life enables Deneris to investigate a series of questions that have been difficult or impossible to address with previous approaches aimed at determining the developmental impact of brain 5 HT synthesis. In Specific Aim 1, Deneris will target Tph2 during fetal life and during the critical early postnatal period to investigate a potential role for 5-HT as an autocrine signal required for homeostatic maintenance of intrinsic 5-HT neuron transcriptional programs, intrinsic 5-HT neuron biochemical and physiological properties and RNA editing patterns. In Specific Aim 2, Deneris seeks to initiate a novel study of the previously unexplored serotonergic epigenome and determine how it impacts stress-related behaviors. Tph2 targeted mice will be used to determine the impact of early-life 5-HT on long lasting programming of histone acetylation/methylation marks and DNA promoter methylation patterns in serotonergic genes and serotonergic histone deacetylase (HDAC) expression. 5-HT neuron-type targeting of HDAC2, an HDAC strongly expressed in developing 5-HT neurons, will be used to determine how alterations in the serotonergic epigenome impacts 5-HT neuron function and stress-related behaviors. In Specific Aim 3, Deneris' team will utilize his powerful 5-HT neuron-type genetic strategies to directly test the long standing hypothesis that 5 HT produced in the brain is a developmental transducer of early life experience and is required to epigenetically program development of the HPA axis and protect against the effects of early life stress.

Precise spatiotemporal control of gene expression is crucial throughout life for maturation of postmitotic neuronal function and preservation of brain health. Although continuously expressed neuronal transcription factors termed terminal selectors, such as Pet1 and Lxm1b, are key regulators of gene expression across fetal and postnatal stages of life the underlying molecular mechanisms through which they control stage specific neuronal gene expression are poorly understood. Here, we aim to fill this gap by comprehensively investigating terminal selector function in postmitotic serotonin (5-HT) neurons. Pet1 and Lmx1b?s control of serotonergic gene expression is of broad interest as 5-HT has wide-ranging modulatory effects on central neural circuitry and altered serotonergic gene expression has been implicated in several developmental neuropsychiatric disorders including depression, stress-related anxiety, autism, OCD, and schizophrenia. The studies proposed here are motivated, in part, by our discovery of changing dependencies of continuously expressed 5-HT neuron terminal effector genes on Pet1 as 5-HT neurons mature. We unexpectedly found that Pet1?s terminal selector control of 5-HT synthesis genes is largely switched off in the early postnatal period and instead Pet1 switches to controlling the upregulation of neurotransmitter GPCR genes, Htr1a, Adra1b, that are needed for afferent synaptic modulation of 5-HT neuron excitability. These recent observations have led us to suggest a new principle that we have termed ?terminal selector target switching?. We suggest that as postmitotic neurons progress through life, continuous terminal selector regulated transcription is not static, as is the prevailing assumption. Instead, we hypothesize that terminal selector regulated transcription is highly dynamic in which regulatory factor interactions with target genes are remodeled as postmitotic neurons mature. We hypothesize the remodeling of regulatory interactions is rooted in the temporal remodeling of postmitotic neuronal chromatin architecture. In Aim 1, we will investigate Lmx1b?s control of early postnatal 5-HT gene expression to determine whether postmitotic target switching is a general property of 5-HT terminal selectors. In Aim 2, we will use our newly developed 5HT-ATAC-seq protocol to uncover the temporal dynamics of 5-HT open chromatin as 5-HT neurons develop and mature from fetal to early postnatal stages of life. Specifically, we will address these questions: Do open chromatin states undergo maturational changes in parallel with the maturation of 5-HT neuron transcriptomes? Do switches in target dependence result from gene specific changes in open chromatin? Aim 3 will investigate a plausible potential mechanism through which developing 5-HT gene expression patterns are controlled: Terminal selectors, Pet1 and Lmx1b, directly act to control the maturation of 5-HT neuronal open chromatin states. Understanding how terminal selectors control postmitotic neuronal gene expression may illuminate potential neurodevelopmental disease mechanisms that cause aberrant neuronal gene expression and defects in neuronal maturation.