Nigel Atkinson - US grants

Nervous systems encode and transmit information in the form of electrical signals. At a cellular level, electrical impulses are generated and modulated by a class of proteins that form ion channels. One class of ion channel proteins that form calcium- activated potassium channels, will be studied. The PI has cloned the gene, slowpoke, that codes for these channels in the fruit fly. A number of different mRNA transcripts that code for slightly different proteins are coded for by this gene. Using molecular techniques, a high resolution map of the tissue and developmental expression of each of the gene products of slowpoke in the nervous system of the fruit fly will be generated. This will be correlated with the biophysical properties of the channels produced. In addition, mutations in slowpoke which result in a unique 'sticky feet' phenotypic behavior will be used to develop genetic screens to identify other genes that control slowpoke gene expression. These studies promise to provide important information on the molecular signals controlling expression of proteins that form ion channels in nerve cells. Such information will be relevant to understanding development and function in the nervous systems of both invertebrates and vertebrates, including human.

ABSTRACT 9724088 ATKINSON Different types of neurons must have different patterns of electical activity for the nervous system to function properly. For example, neurons that initiate movement often "fire" with high-frequency burst of action potentials, whereas neurons that control slow, rythmic behaviors may fire only slowly, but with very precise timing. A neuron's ability to generate specific activity patterns is determined by the types and numbers of ion channels it expresses. Dr. Atkinson and his students will investigate the transcriptional regulation of an ion channel gene in the fruitfly Drosophila melanogaster. The gene, called "slowpoke," produces a calcium-activated potassium channel, which is crucially involved in setting activity patterns in a variety of neurons types. The expression of any gene is controlled by specific areas of the gene called the transciptional control region. In the case of slowpoke, there are four different promoter sequences within the transciptional control region that are responsible for differential expression in different tissues. Some of the promoters alter the amount of the gene product, whereas others alter the amino acid sequence, and thus the structure, of the encoded channel. Such changes can dramatically alter a neuron's range of firing patterns. Dr. Atkinson's laboratory has cloned the entire transcriptional control region of slowpoke. They now plan to systematically introduce deletions into the region to precisely locate the transcriptional control elements responsible for expression in different neuronal subtypes. The function of these control elements will then be verified by re-introducing them as single oligonucleotide sequences, which should restore the expression pattern, if the elements have been properly identified. In the final part of the project, they will use several techniques to identify genes that produce the transcription factors that bind each of the transcription control element. The results of this work will help us understand how neurons acquire the sometimes subtle differences in ion channel number and type they need to play different functional roles.

DESCRIPTION (provided by applicant): We are using Drosophila melanogaster as a model system to understand the acute side-effects of exposure to organic solvent inhaLants. These inhalants, which are abused by a startling large number of adolescents, are commonly found components of many household cleaning solutions and fuels. In general, solvent inhalants owe much of their ability to intoxicate on their anesthetic-like qualities. Abuse of these compounds can have severe side effects. We have observed that Drosophila knocked out by low level exposure to benzyl alcohol vapor recover in approximately 6 hours. This occurs even though one can demonstrate that the solvent in the exposure chamber can still knock out naive flies. Furthermore, the acutely-resistant animals show cross-resistance to the abused solvent toluene. Concomitant with recovery we have observed that mANA from the slowpoke Ca2+-activated K+ channel gene increases in abundance. Because of our many mutant animals and animals carrying various transgenes we will be able to determme the biological meaning of these observations. SpecificalLy, we will determine if cross-resistance and slowpoke mRNA induction is a response common to different abused solvents. During solvent exposure the animals first become hyperexcitable and then 'unconscious'. We would like to know, to which phase, the acute resistance and channel mRNA induction is a response. Using a temperature sensitive mutation in a sodium channel gene which reversibly blocks neural signaling we can answer this question. We will 'turn off' neural signaling during each phase of solvent exposure and determine if the solvent-induced changes occur. Furthermore, using slowpoke mutants we will determine if the change in slowpoke expression is involved in the recovery from solvent-anesthesia. Using our large bank of slowpoke transgenes, which have small lesions in their transcriptional control regions, we will determine if the mRNA abundance change is the result of changed transcriptional regulation and if it results in increases in the channel protein. Finally, we will determine if other ion channel genes shows the same response. We have already shown that a sodium channel gene does not. Future directions include a mutagenic screen to isolate mutations that interfere with the acquisition of acute resistance.

PI: Nigel Atkinson

Abstract
Different types of neurons must have different patterns of electrical activity for the nervous system to function properly. For example, neurons that initiate movement often "fire" high frequency bursts of action potentials, whereas neurons that control slow, rhythmic behaviors may fire only slowly, but with very precise timing. A neuron's ability to generate specific activity patterns is determined by the types and numbers of ion channels it expresses. Nerve cells pick and choose which channels to express by transcriptional regulation. Our understanding of how cells make this decision is very immature. Furthermore, our ability to inspect the sequence of a gene and to then predict its expression pattern is nonexistent. Presently, the purpose of DNA sequences that regulate gene expression (control elements) can only be empirically ascertained. Dr. Atkinson and his students will utilize the genome project, evolutionary studies and functional testing to determine the grammar of regulatory sequences that control ion channel gene expression.
Using Drosophila melanogaster as a model system, Dr. Atkinson has had substantial success describing how the slowpoke Ca2+-activated K+ channel gene is transcriptionally regulated. It has an extremely complex control region. The Atkinson lab has described control elements that specify the developmental- and tissue-specific expression pattern. Most notable was the identification of control elements that differentially activate one promoter in four different muscle subtypes and the identification of an intronic region that modulates developmental specificity.
In the this project Dr. Atkinson will add the Shaker gene to their list of channel genes to be studied. Shaker encodes a voltage-gated K+ channel. Work on slowpoke will also continue. Work on these to genes will enable a comparison of how genes encoding two major classes of K+ channels, the voltage-gated and Ca2+-gated K+ channels, regulate gene expression. In all cases, the focus will be on the regulation of the genes in the nervous system. Transcriptional start sites (~promoters) will be physically mapped and their expression pattern determined using transgenic animals. Because the entire fly is the expression system, the group can study expression in all tissues and organs in their natural developmental context. To identify the control elements the group will make use of the fact that important DNA sequences tend to be conserved over evolutionary time. For each gene, the transcriptional control regions from four different insect species will be sequenced and compared. Small sequences conserved in both sequence and position will be assumed to be control elements. The candidate elements will be tested for function by deleting them from a transgene and then asking how expression had been altered. These methods have been very successful in dissecting the regulation of the slowpoke gene. Furthermore, it will be determined whether the slowpoke and Shaker K+ channels genes show evidence of coordinate regulation; that is, do some of the same combinations of control elements regulate their expression. Because each control element is recognized by a specific transcription factor(s), it will be possible to infer which transcription factors are involved.
Drosophila have the same families of ion channel genes found in vertebrates. In addition, most of the important regulatory cascades affecting developmental gene expression were originally discovered in or shown to exist in Drosophila. Therefore, it is expected that the description of the regulation of channel gene expression in Drosophila will also be relevant to the understanding of the same process in other organisms.

DESCRIPTION (provided by applicant): We are developing Drosophila melanogaster as a model system to understand the molecular basis of rapid ethanol tolerance. Sedating Drosophila for 15 minutes with ethanol causes detectable ethanol tolerance 24 hours later. Concomitant with tolerance, we observe an increase in expression from a Ca2+-activated K+ channel gene. This channel gene is called slo. Mutations in slo prevent the appearance of tolerance, while artificially increasing expression produces resistance. In addition, we observed that reduced expression of slo is associated with ethanol sensitization. Our data indicate that there is a causal relationship between the level of slo expression and tolerance and sensitization. It also strongly suggests that increased slo expression enhances neural activity and that decreased slo expression depresses neural activity. To test this hypothesis, inducible transgenes that express slo channels will be used to produce adult animals that differ in their expression level of the gene. An electrophysiological assay will be used to determine if increased expression is associated with increased neural activity. A measure of behavioral activity will also be used to determine if increased slo expression results in increased behavioral activity. We have observed that during ethanol sedation the overall spike frequency in the brain is reduced. We will determine if the increase in slo expression is a direct response to this reduction in reduced neural activity. Furthermore, we will determine if a transitory reduction in neural activity, by itself, gives rise to measurable alcohol resistance. To do so, we will use a Drosophila mutation that causes reduced neural activity in a temperature-dependent and reversible manner. With this mutation, a temperature pulse will be used to reduce neural activity. The following day, we will determine if the expression level of the slo gene has increased and we will determine if the animals have acquired alcohol resistance.

[unreadable] DESCRIPTION (provided by applicant): We are using Drosophila melanogaster as a model system to understand the acute neural side effects of exposure to organic solvent inhalants. These inhalants, which are abused by a startling large number of adolescents, are commonly found in household cleaning solutions. We observed that a single solvent sedation causes changes in the expression of the slowpoke K+ channel gene and also induces solvent tolerance. We have demonstrated linkage between these phenomena. A mutation that eliminates slowpoke expression also prevents the acquisition of tolerance while transgenic slowpoke expression causes solvent resistance. Our interest is in the mechanics of how nerve cells activate the channel gene expression in response to solvent sedation. We postulate that gene induction induced by this drug experience might involve so-called "epigenetic changes". We will catalog the epigenetic changes induced by sedation across the slowpoke promoter region and correlate these with behavioral tolerance using the chromatin immunoprecipitation assay. We have shown that sedation quickly produces an increase in CREB transcription factor binding at a conserved DNA element in the slowpoke promoter region followed by a spike of histone acetylation at this site. We will use tools unique to Drosophila to specifically block histone acetylation at this site to determine if it simultaneously blocks slowpoke induction and behavioral drug tolerance. Briefly, transposons carrying Gal4 UAS binding sites have been positioned within the slowpoke control region. A chimeric transcription factor made from a Gal4 DNA binding domain and a histone deacetylase will be used to suppress the sedation-induced acetylation spikes within the slowpoke control region. We will unambiguously test the cause and effect relationship between sedation, acetylation of these sites, gene induction and behavioral tolerance. In a related vein small chromosome deletions will be induced in the endogenous slowpoke promoter region using P element mediated mutagenesis (imprecise excision and HEI). These will be used to physically map the site of transcriptional control elements that regulate slowpoke epigenetic changes and gene expression and to determine if the promoter mutations eliminates the capacity for solvent tolerance. We will provide a unified description of how this gene senses and responds to the effects of abused solvent sedation. [unreadable] [unreadable] [unreadable]

Ion channel proteins produce the electrical impulses used to transmit information in the nervous system. Each neuron encodes a large complement of channel genes and the electrical character of the cell is dependent on the subset and quantity of channels that it expresses. In the mature nervous system, the cell must coordinately regulate the activity of ion channel genes to ensure that the properties of the cell do not drift. The Atkinson lab focuses on how channel gene expression is regulated in adult animals. They use the Drosophila model system because of the unusual molecular tools that it provides. The slo gene that they study encodes a BK type Ca2+-activated K+ channel which is highly conserved in structure and function from insects to mammals. The Drosophila slo gene has one of the best described transcriptional control regions of any channel gene. The Atkinson lab has shown that the slo gene responds to aberrant changes in neural excitability in a fashion predicted to restore normal activity. They use organic solvents as anesthetics to reduce neural excitability and observe that the animals up regulate slo gene expression. This change makes the animals resistant to further attempts at sedation (a behavior called tolerance). It is the transcriptional mechanics that link neural excitability to slo gene expression that the lab seeks to determine. The Atkinson lab has long studied the regulation of ion channel gene expression and has the tools and experience to address this question. The first objective is to use mutant analysis to identify transcription factors that up regulate slo after sedation. Flies carrying mutant transcription factor genes are tested to see if the mutation interferes with sedation-induced up regulation of slo. To date, two mutations have been identified and additional reasonable candidates exist. The second objective will be to determine if these factors act by directly by binding the slo promoter region or through an intermediary transcription factor. The chromatin immunoprecipitation assay will be used to see if the protein is physically present on the slo promoter region. Furthermore, by quantifying the amount of bound transcription factor in sedated and mocksedated animals, the lab will determine if sedation increases the binding of the transcription factor to its binding site. The third objective will be to determine the order of action of each transcription factor in the process. Previously, the lab used the chromatin immunoprecipitation assay to describe various epigenetic changes that occur across the slo promoter region following sedation. These changes are thought to be produced by transcription factor activity. Mutations in the responsible transcription factor gene are expected to block specific steps and interrupt the progression of these changes. By determining where each mutation truncates the process, the order of transcription factor activity can be determined. I

This project is designed from the ground up to have aspects that are readily accessible to undergraduate and high school students. This will enable the students the chance to join the lab and to perform important experimental work while they acquire the skills required for the technically difficult molecular portion of the project. Importantly, the undergraduates selected for the project will originate from both U.T. and from the minority institution, Texas A&M Kingsville. This project will be an important tool for encouraging minority participation in the life sciences.

Alcoholism, a disease of changed behavior, is believed to involve alterations in gene expression. The knowledge gap to be filled is that of understanding how ethanol regulates gene expression to produce behaviors associated with alcoholism. It is our long-term goal to understand the transcriptional mechanisms that underlie ethanol-induced changes in gene expression that produce these behaviors. Functional tolerance, a metabolism-independent reduction in ethanol sensitivity due to prior exposure, is a change in behavior that can cause increased alcohol consumption and speed the path to addiction. Experiments in mammals, worms, and insects show that BK channels play an evolutionarily conserved role in ethanol responsivity. The highly conserved slowpoke (slo) gene encodes BK-type Ca2+-activated K+ channels, which integrate Ca2+ signals and electrical signals and are central to modulating neural activity. In Drosophila, ethanol sedation induces slo gene expression, and this expression has been shown to cause functional rapid tolerance to ethanol sedation. It has recently been shown that epigenetic modification to gene promoter regions underlies important aspects of long-term changes in behavior caused by experience and drugs. These modifications are believed to form an extended code that regulates gene expression. Induction of slo expression has been linked to epigenetic modifications of histones across the promoter region of the gene. The central hypothesis, formed from substantial preliminary data, is that ethanol sedation activates transcription factors and produces histone modifications that together result in increased slo expression to produce tolerance. It is the objective of this proposal to identify the transcription factors and epigenetic histone modifications that cause ethanol-induced slo expression. This objective will be achieved by pursuing the following specific aims: 1) determination of the time course of ethanol- induced histone modifications across the slo promoter region; 2) identification of transcription factors that modify histones and induce slo expression in response to ethanol sedation; and 3) identification of the cis-acting enhancers in the slo promoter region that are used in the ethanol-stimulated modification of histones and that are involved in the induction of slo. This work is innovative because it uses the unique toolset of Drosophila to study the epigenetic regulation of a gene known to underlie functional ethanol tolerance. The research is relevant to the mission of NIH/NIAAA because it has the potential to illuminate the fundamental mechanisms underlying functional tolerance and thereby the path to alcohol addiction.

DESCRIPTION (provided by applicant): The potential of genomics to identify genes important for alcoholism has not been completely realized because ethanol produces a surfeit of changes in gene expression, most of which do not seem to make any meaningful contribution to the ethanol response of interest. A simple and effective way to pan for genomic changes relevant for an alcoholism phenotype would be invaluable. The problem is solved by focusing on genes that show common changes produced by two chemically and metabolically dissimilar drugs, ethanol and benzyl alcohol, which produce cross-tolerance by a mechanism with a shared molecular origin. Genes that respond in the same way to both alcohols will be identified by genomically surveying epigenetic histone modifications and gene expression. These will be functionally tested by mutant analysis in the fruit fly Drosophila melanogaster, an animal that has ethanol responses remarkably similar to humans. The alcohol response genes will be tested for a functional role in ethanol tolerance and two withdrawal/dependence symptoms - alcohol withdrawal induced hyperexcitability and cognitive ethanol dependence. Parts of this response have already been shown to be conserved in mammals. To directly bridge our work to human alcoholism the data will be collaboratively compared to human alcoholic datasets. The advantage of using Drosophila is that candidate genes can be rapidly and functionally tested by mutant analysis for their role in producing these ethanol responses. In Aim 1, chromatin and RNA will be prepared from the heads of flies treated with ethanol or benzyl alcohol. Epigenetic and gene expression changes common to both drugs will be identified by ChIP-seq to identify changes in histone H3 and H4 acetylation and methylation (H3K36me1 and H3K36me3). These histone marks have tight linkage to changes in gene activity. RNA-seq will be used to monitor changes in gene expression. These candidates and pre-existing candidates from an epigenetic screen will be tested in Aim 2 and 3 by mutant, RNAi, and mis-expression analysis. In Aim 2, candidate genes will be tested for a role in functional tolerance to ethanol sedation. In Aim 3, th effects of mutations on alcohol-withdrawal will be determined electrophysiologically in adults and behaviorally in larvae. Adult flies show alcohol-withdrawal hyperexcitability that increases the susceptibility for seizures. While in ethanol-adapted larvae, the capacity for associative learning declines during withdrawal and is restored after ethanol reinstatement-demonstrating functional dependence. This contribution will be significant because it will define the origins of functional tolerance phenotype and establish its relationship to dependence phenotypes. The proposed research is innovative because it uses 1) a shared drug response to focus a genomic survey on ethanol-response genes, 2) epigenetic histone modifications to help identify genes that transcriptionally respond to ethanol intoxication, and 3) new behavioral assays that allow us to detect tolerance and physiological dependence in flies. Over its 5 year span, this project will be used to train 2 post-docs, 2 graduate (Ph.D) and 10 undergraduate students.

? DESCRIPTION (provided by applicant): At the core of the behavioral changes characteristic of alcoholism is the rearrangement of gene expression in the brain of an addicted individual. However, there exists a fundamental gap in our understanding of how alcohol consumption produces this rearrangement and leads to subsequent changes in behavior. Previous work in a mammalian system described how a particular microRNA (miRNA) acts as a key intermediary in the production of alcohol-induced changes in gene expression that produce functional-alcohol tolerance. The objective of this application is to combine the power of next generation sequencing with Drosophila genetics to identify all alcohol-sensitive miRNAs and to functionally test them for a role in producing an alcoholism-related behavior: functional ethanol tolerance (inducible ethanol resistance). This research goal stems from collaboration between two labs, one specializing in alcohol regulation of miRNA in the mammalian system, the other being an expert in fruit fly genetics and epigenetic mechanisms of alcohol actions. Based upon preliminary data and review of the literature, the central hypothesis is that even acute ethanol exposure causes long lasting changes in miRNA expression that lead to a rearrangement of the brain transcriptome. To test this hypothesis, miRNA-Seq and RNA-Seq will be performed on flies that have been briefly sedated with ethanol. Three different time points will be assessed in order to capture early and late changes in miRNA expression and their targets. Then, each ethanol-responsive miRNA will be functionally tested to determine if it contributes to functional ethanol tolerance (inducible-ethanol resistance). In flies, ethanol tolerance is a product only of neural adaptation to ethanol. Therefore, we will manipulate miRNA expression levels in the nervous system using the Gal4/UAS system and then determine its effect on functional ethanol tolerance and ethanol resistance. Achieving these goals will help identify and evaluate the role of miRNAs in the neural adaptations that underlie ethanol tolerance and will contribute to our longer-term goal to understand how ethanol consumption modulates gene expression to promote alcoholism.