Vicki L. Chandler - US grants

Understanding how genes are differentially regulated during the development of an organism is a fundamental problem in biology. Genetic studies of maize have provided many examples of programmed changes in gene expression during development and have identified regulatory phenomena such as paramutation and the effects of controlling elements. One such genetically characterized locus in maize is the B locus which regulates the expression of anthocyanin pigments in the plant. Variation in the tissue-specific synthesis of anthocyanins is determined by which allele is present at B. Examining different B alleles at the molecular level should reveal nucleotide sequences responsible for regulating the quantity, timing and tissue-specificity of pigment production. Certain B alleles undergo or promote paramutation: a heritable alteration in gene expression promoted by the presence of two specific alleles in the same plant. Many models have been proposed to explain paramutation, including alteration in chromatin structure, gene conversion, or the interaction or transposition of DNA elements. A molecular description of this allelic interaction should reveal general principles on cell heritable regulation of gene expression as it occurs during development. These principles should be applicable to other organisms where a combined genetic and molecular approach is not yet feasible. The long term goals of this proposal are to examine the molecular mechanisms controlling the expression of the B gene in maize. The specific aim of this proposal is to clone the B genomic sequences using the transposable element Robertson's Mutator to mark the locus. Multiple independent insertion mutations have been isolated in order to increase the probability of obtaining the entire gene. The gene will then be recovered by taking advantage of the homology between the element inserted into the B gene and the cloned transposable element. The mutants and the cloned sequences generated in this study, will provide tools with which to begin a molecular analysis of paramutation and gene expression at B.

Understanding how genes are differentially regulated during the development of an organism is a fundamental problem in biology. Genetic studies of maize have provided many examples of programmed changes in gene expression during development and have identified regulatory phenomena such as the effects of transposable elements on gene expression and paramutation. One such genetically characterized locus in maize is B, which regulates the expression of the purple anthocyanin pigments in the plant. Variation in the developmental timing and tissue-specificity of anthocyanin synthesis is determined by which allele is present at B. Examining different B alleles at the molecular level should reveal nucleotide sequences responsible for regulating the timing and tissue- specificity of pigment production. Certain B alleles undergo or promote paramutation: a heritable alteration in gene expression promoted by the presence of two specific alleles in the same plant. Many models have been proposed to explain paramutation, including gene conversion, alterations in chromatin structure, DNA modification, or the interaction or transposition of DNA elements. A molecular description of this allelic interaction should reveal general principles on cell heritable regulation of gene expression as it occurs during development. These principles should be applicable to other organisms where a combined genetic and molecular approach is not yet feasible. The long term goals are to examine the molecular mechanisms controlling the expression of B. The specific aim of this proposal is to clone the B genomic sequences using transposable elements and to use the cloned sequences and mutants to begin analyzing the structure and function of B. Multiple independent insertion mutants and revertants have been isolated. The mutants and revertants isolated from them, have varied phenotypes with respect to the tissues in which pigment is synthesized. The insertion of an element into B will produce a molecular marker that segregates with the mutant phenotype. The element and the adjacent gene will be recovered by taking advantage of the homology between the element inserted into the B gene and the cloned transposable element. The cloned sequences will then be used to determine the structure and expression of the wild type and mutant B alleles as well as the molecular basis for paramutation.

Our goal is to understand the mechanisms by which the expression of the maize anthocyanin biosynthetic enzymes is coordinately induced by the regulatory protein B. The B gene product is required for the steady state accumulation of mRNA from several genes encoding anthocyanin biosynthetic enzymes (structural genes), and we have recently demonstrated that the B gene plays a major role in stimulating the transcription of the structural genes. Transient cotransformation studies using plasmids carrying structural gene promoter sequences fused to luciferase, and plasmids which produce B protein, demonstrate B mediates an approximately 100 fold induction in luciferase activity. The deduced amino acid sequence of the B protein (obtained from the cDNA sequence) revealed a myc homologous region found in several DNA binding proteins and a negatively charged region characteristic of transcriptional activators. Both in vitro and in vivo assays will be used to: examine the in vivo modification of the B protein and its subcellular location and tissue distribution; dissect the regions of the B protein necessary for transcriptional control of the structural genes; determine the functions of these regions (transcriptional activation, DNA binding, protein/protein interactions, etc.); assess whether the B protein interacts directly with regulatory regions found in the structural genes; identify other proteins interacting with B; and investigate if B influences anthocyanin biosynthesis at levels in addition to transcription of the structural genes. The maize anthocyanin biosynthetic pathway represents one of a small number of pathways in higher plants for which regulatory proteins and their targets are known. Thus, studying the mechanisms utilized by B should reveal important information regarding regulated gene expression in higher plants.

Extensive genetic analyses of the maize anthocyanin pathway have revealed multiple target genes, whose expression is coordinately controlled by several regulatory genes. Our goal is to understand the mechanisms by which the transcription of the genes encoding the anthocyanin biosynthetic enzymes is coordinately controlled by three regulatory genes, b, c1, and a3. The proposed in vivo and in vitro experiments should enable us to begin to distinguish between possible models for how the regulatory proteins are interacting to control transcription. Our specific aims are to : 1) mutationally dissect the regions of C1 and B required for their interaction, 2) identify other proteins involved, 3) develop in vitro assays for analyzing B and C1 protein/protein interactions, 4) assess whether the B and C1 proteins interact directly with structural gene regulatory sites, 5) identify the cis-acting sites required for B and C1 regulation of uncharacterized structural gene promoters, 6) identify functionally important regions of B using available transposable element insertions, 7) determine if a3 is acting at the transcriptional level and if so, clone and characterize a3. %%% Principles of gene regulation learned from these studies should apply to other systems where a combined genetic and molecular approach is not yet feasible.

9413223 Lynch This award renews support of a joint training effort by 14 faculty who are members of the Department of Biology and who belong to one of three interdepartmental units (the Institutes of Molecular Biology, Neuroscience and Marine Biology) or to the Ecology and Evolution Program. The theme of this RTG is molecular evolutionary biology. The research and training are broadly focused on experimental and theoretical study of genetic mechanisms, developmental biology and population biology, all considered at the molecular level and all approached from an evolutionary standpoint. The research programs of the faculty involve a diverse set of organisms including bacterial viruses, fungi, higher plants, drosophila, zebrafish, and other invertebrate and vertebrate animals. Training activities involve students at the undergraduate, graduate and postdoctoral levels. Undergraduates participate during both academic year and the summer term, generally during their junior and senior years. Graduate student participants are recruited from first year students in any of the institutes or programs listed above. Postdoctoral fellows are recruited through national advertising. Besides research, training include an RTG colloquium featuring about 10 outside speakers each year, a journal club and a set of 4 courses in each of three areas: evolutionary, developmental and molecular biology. Of the 12 courses, 5 were developed for the RTG. Graduate trainees are required to take 5 courses, including at least one course in each area. All trainees are required to participate in the journal club and colloquium. In addition to these, a new program of 3 day-long mini-symposia per year will begin in the renewal period. Each symposium will involve 3 - 5 outside speakers and be preceded by a 3-4 week journal club focused on the topic of the symposium. ***

Abstract Not Provided

[unreadable] DESCRIPTION (provided by applicant): The ability to combat disease depends to a great extent on a solid understanding of how gene expression drives development and maintains life. To this end, the compelling primary goal of the proposed conference is to clarify the manner in which chromatin controls gene expression, both at the level of the single gene and at the level of the whole genome. The conference will constitute the 7th meeting of the biennial FASEB Summer Research Conference on Chromatin and Transcription, a series that, from its inception, has been internationally recognized as one of the two most important meetings in the field, the other being its sister Gordon Conference held in alternate years. Thus far, 100% of 27 respondents have agreed to attend, each one an undisputed leader in his or her field. As such, there is no doubt that this conference, like its predecessors, will host important advances in gene regulation. [unreadable] [unreadable] In order to maximize the success of the conference, the co-organizers are committed to 1) bringing in experts from diverse fields of chromatin, transcription, and genome organization, so that the conference can be strengthened by synergy between researchers who would not normally meet, 2) introducing new issues and raising new questions, and 3) assuring the future of the field by promoting the careers of young investigators. [unreadable] [unreadable] At present the program will consist of nine platform and two poster sessions, and total attendance is expected to be ~200, including ~60 speakers. While the program is expected to evolve, the session titles are, at present: [unreadable] [unreadable] I. Histone Modification [unreadable] II. Nucleosomal structure and remodeling [unreadable] III. Replication, assembly, repair, maintenance, and segregation [unreadable] IV. Higher order structure and regulation [unreadable] V. Non-coding RNAs and regulation [unreadable] VI. Developmental roles of chromatin structure [unreadable] VII. Higher order effects and development [unreadable] VIII. Transcription I [unreadable] IX. Transcription II [unreadable] [unreadable] [unreadable]

Overview: I am nominating myself for a NIH Director[unreadable]s Pioneer Award, which if awarded, I would use to investigate homology-dependent epigenetic mechanisms of gene regulation in animal models and eventually humans. One aspect of my research has been to study a phenomenon called paramutation, which was discovered in plants and involves allele communication that leads to a mitotically and meiotically heritable change in gene expression. My hypothesis is that the paradigm-shifting mechanisms of allele communication, which we have uncovered operating in plant chromatin, also exists in mammals and could explain the aberrant segregation of certain [unreadable]genetic[unreadable] diseases. I have organized this essay in 3 sections. In Background and Significance, I define epigenetics, briefly summarize the fields[unreadable] current understanding of mechanisms and discuss its importance for understanding a number of human diseases. In Prior and Current Work, I summarize our prior work on paramutation, using this as a basis for illustrating my approach to science providing evidence for why this nomination should be considered in the NDPA process. In New Research Direction, I briefly describe the new approaches I am proposing, explain how this builds on my past work, and discuss why the NDPA process provides a unique opportunity to pursue this work.