Douglas L. Chalker - US grants

The ciliated protozoan Tetrahymena thermophila excises an estimated 6000 specific DNA segments from its somatic nucleus during development. This project aims to understand the regulation of this massive genome reorganization and ultimately learn fundamental principles governing chromosome structure and genetic stability. Understanding how particular DNA segments, called deletion elements, are selectively excised is challenged by the fact that they are quite diverse in size and sequence. The PI's approach toward understanding how the cell recognizes these diverse sequences has been to understand in detail the mechanism of deletion of two DNA segments, called the M and R deletion elements, and thus identify common regulatory properties. In this work, these elements will be mutagenized in order to identify the specific sequences that target these DNA segments for elimination. Mutant deletion elements will be integrated into the genome such that their excision can be monitored during the process. This will allow the determination of mutations in the identified cis-acting sequences that abolish the association between the element and proteins involved in excision. A major goal of these mutational analyses is to delimit the sequences that activate the non-genic transcription of deletion elements and determine whether transcription is required for DNA deletion. To further explore the molecular basis of epigenetic regulation of these DNA rearrangements, the minimal requirements for this regulation will be defined. Several prior genetic studies support a model that RNA copies of the element produced from parental nuclei act as inhibitory signals that block DNA deletion of the homologous element. This and other possible explanations will be tested. Combining studies that address both genetic and epigenetic regulatory components will expedite a comprehensive understanding of this process.

This project investigates a process of regulated DNA rearrangement that occurs during development of the ciliated protozoan Tetrahymena thermophila. The cellular machinery that accurately rearranges the Tetrahymena genome likely involves many of the components that ensure the proper genetic stability of this organism. This research aims to understand how particular DNA segments are targeted for elimination. Its long-term goal is to learn how removal of these DNA segments affects the normal genetic stability of the somatic genome. Investigation of this process in Tetrahymena should provide fundamental insight into genetic mechanisms that operate in all organisms.

DESCRIPTION (provided by applicant): The ciliate Tetrahymena thermophila excises approximately 6000 specific DNA segments from its somatic nucleus during development. This project aims to understand the regulation of this massive genome reorganization and ultimately learn fundamental principles governing chromosome structure and stability. Understanding how particular DNA segments, called internal eliminated sequences (IES), are selectively excised is challenged by the fact that they share little common structure. Currently, we have limited knowledge of the protein machinery that recognizes and excises these 6000 IES. While four proteins have been linked to this process, their exact roles have yet to be fully elucidated. We have identified five genes encoding novel proteins putatively involved in DNA rearrangement. These were identified using a Green Fluorescence Protein (GFP)-based screening strategy designed to find developmentally expressed proteins that localize specifically to the differentiating nuclei where and when DNA rearrangement occurs. To demonstrate whether these five candidates are required for rearrangement, we will knock out their genes and analyze the phenotype of the resulting transgenic strains. Furthermore, we will test whether these proteins interact with previously identified DNA rearrangement proteins and/or associate with the IES during their excision. Initial characterization of one of these genes, LIA1, indicates that its protein very likely participates in DNA rearrangement. Characterization of additional components of the DNA rearrangement machinery will greatly enhance our understanding of this process. This biological phenomenon provides a unique system with which to discover mechanisms cells use to recognize individual chromosomal segments throughout the genome. Such mechanisms are likely critical for ensuring chromosome stability. Given that cancer cells commonly exhibit aberrant DNA rearrangements, identification of the cellular mechanisms that ensure faithful chromosome maintenance is an important step leading to an understanding of the molecular basis of disease associated with genetic instability.

RNA interference (RNAi)-related mechanisms participate in diverse epigenetic phenomena. Few are more extreme than the genome remodeling of the ciliate Tetrahymena thermophila. This organism eliminates nearly 15 megabases of its germline DNA from the somatic nucleus during its development. This project aims to understand the regulation of this massive genome reorganization and ultimately learn fundamental principles governing chromosome structure and stability. Understanding how ~6000 DNA segments, called internal eliminated sequences (IESs), are selectively excised is challenged by the fact that they share little common structure. The current model is built on the observations that bi-directional IES transcription leads to the generation of ~28 nucleotide RNA molecules (scan RNAs) that then target specific modification(s) to the homologous chromosomal location(s). The DNA rearrangement machinery recognizes this modified chromatin state and eliminates the targeted DNA segment. These studies will certainly provide fundamental insight into RNAi-related mechanisms that direct chromatin modifications that are critical for transcriptional gene silencing and heterochromatin formation in eukaryotes. The specific intellectual goals of this project are: 1) Identification of general cis-requirements for IES excision that should define basic constraints imposed on RNAi-directed chromatin modification. 2) Characterization of the specificity of germline, non-genic transcription that is postulated to be the initiating event in this remarkable genome reorganization. 3) Continued characterization of Dicer-like, RNAseIII homologues of Tetrahymena and their putative partner proteins. This will lead to elucidation of their roles in DNA rearrangement and/or other RNAi-related processes. The plan is to accomplish these goals through a combination of genetic and molecular biological approaches, taking specific advantage of tools available for studies in this model organism. Underlying this project is a goal to understand how RNA molecules can communicate genetic information between the parental and developing genomes, which has great potential to reveal novel roles for RNA in epigenetic programming. Additionally, because it is believed that many of the DNA segments targeted for elimination are important for germline chromosome structure, an increased understanding of how the cell specifically recognizes these sequences will contribute to the current knowledge base of mechanisms that ensure the chromosome stability that is essential to prevent aberrant rearrangements.

The ability of RNAs with sequences complementary to genomic DNA to regulate the activity of the eukaryotic genome has been revealed by recent studies of diverse biological processes, including genome rearrangements of Tetrahymena. The unique biology of this organism offers an excellent context which with to uncover the fundamental mechanisms by which such RNAs elicit their action on the DNA. In addition to providing such insights into this important genetic regulatory mechanism, this project will serve to train undergraduates, post-baccalaureate laboratory technicians, and PhD students in hypothesis-driven research and prepare them for future scientific careers. Underrepresented minorities in science have been trained using prior support and this continued support enables future mentoring. The research is directly linked to the further development of a laboratory course in which undergraduate students engage in original, sophisticated research. This course's problem-based approach teaches students how to use current technologies in a model organism to generate a thorough understanding of the biological process being explored. Gene silencing vectors that will be developed in this project will be incorporated into the design of future offerings of this course, providing a broader impact of the project's intellectual pursuits allowing direct carryover into its educational goals. The research tools and curriculum generated by this project, such as these vectors for gene inhibition studies, will be extremely valuable reagents that will be distributed to the broader Tetrahymena research and education community. Thus these efforts will help make this important model organism more accessible to biologists from other fields.

Engaging more undergraduates in research experiences is a priority for improving science education and course-based research experiences are a promising approach to reaching larger numbers of students. The Ciliate Genomics Consortium (CGC) is a student-centered, nation-wide collaborative learning community that uses scalable functional genomics research for integration into courses in a variety of biology sub-disciplines. The CGC employs an integrative teaching and research model that combines both inquiry-driven class laboratory activities and collaborative consortium pedagogies to advance faculty research. Previously, the CGC developed modular course-based research curricula that, when adopted by the research community using the ciliate Tetrahymena, effectively engaged greater numbers of students in authentic research while advancing faculty research. This work expands the consortium by creating new or improving tested curricula to promote their broad adoption, creating more opportunities for teaching/research integration. If successful, this project provide evidence that students in classroom settings can contribute substantially to faculty and community research priorities with a variety of model organisms.

To achieve project goals, the CGC will: 1) develop curricula adaptable to faculty research interests, integrate consortium activities with research community resources, and assess student learning gains; and 2) disseminate the CGC model through training workshops and assess the impact on faculty teaching and student learning. Curricula are disseminated through annual workshops and test whether research communities can foster learning communities that promote faculty adoption of classroom-based research as a high impact teaching practice. In this model, members of a research community form a professional learning community to enhance and apply best STEM teaching practices. To learn more about the effectiveness of this approach for both students and educators, the project will assess the pedagogy and report any conceptual gains this research-based curriculum offers over other instructional models, and present limitations and challenges observed. Several validated instruments will be used to measure confidence and learning gains with newly developed assessments to evaluate predicted cognitive gains. Cohorts of students at each institution are identified, not engaged in the CGC curriculum, to control for instructor and institutional factors.

The genomes of many organisms contain DNA with no known function. In some organisms, this so-called junk DNA is eliminated during development by a complex rearrangement of the genome. However, it is not clear how the cell can tell what is junk and what is not. This research will study how the ciliate, Tetrahymena thermophila, establishes what parts of the genome should be thrown away as junk and what should be retained as functionally important. Because junk DNA is found in the genomes of many organisms, this work could have broad scientific significance on understanding how cells deal with extraneous, non-essential DNA. To accomplish the research, a tool for visualizing genome-related datasets will be created and made accessible for use by the broader scientific community, thereby improving the current scientific infrastructure. The project will enhance the future scientific workforce by engaging trainees from the undergraduate to post-doctoral level in hypothesis-driven research. The research efforts will be coordinated with curricular development activities of the principle investigator, and thus will facilitate best teaching practice by bringing authentic research into the university biology classroom. Students engaged in the classroom research, in turn, will gain first-hand experience in the practice of scientific discovery and add to a growing body of scientific knowledge.

This project will elucidate mechanisms that eukaryotic cells use to establish boundaries between functionally distinct regions of the genome by exploiting the model experimental organism, Tetrahymena thermophila, which eliminates much of its non-coding DNA from the somatic copy of its genome. Using a combination of genetic and biochemical approaches, the work will uncover mechanisms that Tetrahymena cells use to define the boundaries of the DNA segments that are eliminated during its development. The fact that Tetrahymena eliminates these sequences allows one to unambiguously identify those that get packaged as junk by comparing the content of the reorganized somatic genome and the intact germline genome. The project will use available Tetrahymena genome sequences to generate a genome browser that will map the retained and eliminated regions, using the intact genome as a reference. Previous research revealed that a regulatory protein called Lia3 is critical for the accurate removal of a subset of the eliminated sequences. The genome browser will be used to compare the DNA of normal cells to DNA from cells lacking LIA3 to identify all the sequences for which elimination boundaries are inaccurately defined when this protein is absent. Preliminary experiments have revealed that the sequences controlled by Lia3 contain guanine (G)-rich sequences positioned about 50 base pairs from each boundary. Lia3 binds these sequences when they form G-quadruplex DNA, a four-stranded DNA structure. This represents a novel DNA binding activity, and the project will thoroughly characterize both the ability of Lia3 to bind this non-standard DNA structure and elucidate how this structure can serve to organize the genome. In addition, the Tetrahymena genome encodes three other proteins that are similar to Lia3 in sequence and gene expression, and the project will test the hypothesis that each of these proteins identifies the boundaries of a distinct subset of eliminated DNA. Together, these approaches will characterize a previously unknown mechanism that eukaryotic cells use to define boundaries between genes and non-coding DNA and provide clear evidence that G-quadruplex structures have important regulatory roles.

The genomics revolution, the wide-spread use of DNA sequence data to enhance biological understanding, brings unique challenges for biology educators. Skills from computer and data sciences have become core competencies for students even though many life science faculty completed formal training without the computational expertise needed to effectively engage in this field. The Genomics Education Alliance (GEA) will bring together members of existing genomics education networks, leveraging their combined expertise to identify and curate common genome analysis tools, associated curricular and assessment materials, and faculty training strategies to facilitate the adoption of genomics instruction at any college or university. By making existing resources accessible, the GEA will enable current faculty to guide undergraduate life science students participating in authentic genomic research projects. Such work will enhance the ability of students -- whether at 2- or 4-year institutions -- to become productive members of the technological work-force, to succeed in post-graduation studies in biology and related disciplines, and to be better informed citizens and decision makers. The GEA will develop a consensus set of vetted resources and training materials to be disseminated to the education community through a single web portal for use of these materials in undergraduate classrooms. Curation efforts will emphasize faculty training and student learning resources, in particular the development of strategies to effectively lead genomics Course-based Undergraduate Research Experiences (CUREs).

The GEA will form an extended network, recruiting members from existing undergraduate genomics education communities, and exploit their combined experience to create a unified and sustainable platform, available through a web portal, that faculty can use to engage diverse students in inquiry-based curricula and CUREs. Our efforts will focus around three major aims: 1) curation and maintenance of computational resources for genomics instruction; 2) development of faculty training and student learning materials; and 3) evaluation of teaching and learning assessments in genomics education, aligning assessments with faculty training and student learning resources. The GEA network will be organized into four committees -- Bioinformatics Infrastructure, Student Learning, Faculty Training, and Evaluation/Assessment -- who will meet regularly to assemble and select materials to be further refined at the three face-to-face workshops. The specific tasks of each committee will be set at the kickoff workshop, and their initial work will be reported at a second progress workshop. At the final synthesis workshop, participants will evaluate the products and recommend any continued development needed for broad implementation. Curated materials will be maintained on established infrastructure to ensure the long-term accessibility of these products.

This project is being jointly funded by the Directorate for Biological Sciences, Division of Biological Infrastructure, and the Directorate for Education and Human Resources, Division of Undergraduate Education as part of their efforts to address the challenges posed in Vision and Change in Undergraduate Biology Education: A Call to Action (http://visionandchange/finalreport/).

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary / Abstract This proposal seeks continued funding for the Tetrahymena Stock Center in order to maintain its current operations, expand its capabilities and ensure its sustainability as a resource for the community at-large. A key model for eukaryotic cell and molecular biology, Tetrahymena thermophila has been instrumental to our understanding of a wide range of biological phenomena with direct relevance to human health and disease including cancer, infertility, aging, respiratory and neuroendocrine dysfunction. Tetrahymena has also shown great promise as a platform for the production of recombinant proteins, including vaccine antigens and difficult- to-express human ion channels, and serves as a useful teaching tool in K12 and undergraduate classrooms. Specifically, our aims for the Resource are to 1) continue to act as a strain repository accepting new strains and making live cultures of T. thermophila available to interested users at reasonable cost; 2) complete the annotation of archival strains currently housed at the Center and acquire next-generation cloning vectors for micro- and macronuclear genome-editing; 3) offer expanded services and develop innovative marketing approaches to increase revenues. Additionally, to ensure insure long-term stability of the resource we plan to 4) relocate the repository from Cornell University to Washington University in St. Louis; 5) migrate all data from the legacy Tetrahymena Genome Database (TGD) schema to CHADO and transfer its web-hosting functions from Bradley University to the NSF-funded National Center for Genome Analysis Support (NCGAS) at Indiana University; and, 6) redesign the TGD website to display relevant data via the Tripal framework. Our additional Research aims are to: 7) develop genetic strains lacking a functional ribosomal DNA locus to enable selection-free creation of new cell lines; and, 8) develop CRISPR-mediated genome editing tools to enhance the overall utility of the Tetrahymena platform.

Project Summary The Genomics Education Partnership (GEP) is a nationwide faculty-driven collaboration that, through training, mentorship, and outreach enables a broad range of institutions to introduce bioinformatics into the undergraduate curriculum. Bioinformatics training extends the teaching of molecular biology, strengthens students' computer science and math skills, and emphasizes the power of computational approaches to explore biological systems. Inquiry-driven genomics research engages students in scientific discovery while maintaining a widely distributed network of teacher-scientists proficient in cutting edge experimental techniques. To date the GEP has trained hundreds of faculty and impacted thousands of undergraduates. A majority of participating faculty and students are women, and a third of GEP participant institutions are minority-serving. From the start, the GEP has sought to: 1. Introduce bioinformatics into the undergraduate curriculum through research 2. Create a scalable system to tackle big projects with many students working in parallel 3. Model ?team science? through collaboration of a widely dispersed team 4. Publish results in the scientific literature with faculty and student authors co-authors 5. Publish assessment results to contribute to the scholarship of teaching and learning. The GEP engages undergraduates in meaningful genomics research regardless of the selectivity, location, or research focus of their institution, as supported by our published assessments of education outcomes. This IPERT proposal will further enhance the GEP's commitment to inclusive training of the scientific workforce. Specific Aim 1 will develop regional nodes to recruit, train, and mentor inclusive local communities of faculty and students engaged in genomics teaching and research. New faculty recruitment locally and at equity-promoting national STEM conferences will focus on institutions that serve students from underrepresented groups. Regional symposia will enable undergraduate students' direct dissemination of their research and participation in the scientific community. Specific Aim 2 will broaden the GEP's scientific scope, capacity, and dissemination by creating a new investigator-initiated, undergraduate-powered gene annotation workflow, with an associated genomics curriculum, that will enable community annotation projects for any eukaryotic genome, driving scalable team science approaches to novel and emerging genomics questions. This new platform incorporates rapid open-access micropublication of gene reports by undergraduate authors, further engaging students in their science.

Project Summary/Abstract This proposal builds on an existing infrastructure of the ASSET program (Advancing Secondary Science Education through Tetrahymena), to generate new teaching materials, reach new student populations, and ensure sustainability of the program by transitioning its overall functions from Cornell University in Ithaca, New York to Washington University in St. Louis. Over the past 10 years, ASSET has built a highly successful SEPA program that teaches core biology content to primary, middle, and high school students using a safe, easily grown, and behaviorally complex single-celled organism (viz. Tetrahymena). Tetrahymena provides an ideal platform for teaching basic principles of cell structure and function, genetics, evolution, sex, prey-predator interaction, cell signaling, etc. without engendering in students any of the conflicting reactions often evoked using live vertebrate animals. Additionally, Tetrahymena offers a graphic illustration of the deleterious effects of toxic and/or addictive substances on living cells in real-time, equipping teachers with a powerful tool with which to fight against substance abuse and promote healthy behavior. ASSET provides stand-alone laboratory kits that are easily integrated into existing science/health curricula, along with innovative co-curricular modules that address the intersection between science and society. The program helps science teachers educate students in under-resourced schools in rural and inner-city school districts, provides robust on-site and distance teacher development activities, while continuously being evaluated for pedagogical effectiveness. This new proposal will greatly expand the program?s current offerings by introducing new materials to existing modules, as well as new modules that address recently identified areas of high programmatic interest to SEPA, specifically, embedding math in P-8 teaching projects; exposing students to research-generated data; and, training students in informatics, bioinformatics, and data science. The Co-Directors have extensive experience teaching bioinformatics and helping students interpret research-driven data, while curriculum specialists at Washington University?s Institute for School Partnership are well-positioned to evaluate existing modules to identify opportunities to teach mathematical concepts using examples from biology and student generated data at grade appropriate levels. Finally, the move from Cornell to Washington University addresses an additional area of programmatic interest for SEPA, namely, adapting successful SEPA programs to new areas or with new populations. Through its Institute for School Partnership, Washington University is strongly committed to achieving equity in K-12 education bringing high-quality STEM teaching to >100,000 students in the Midwest through its various teaching programs. Incorporating the ASSET program under its umbrella expands its current activities, introduces ASSET to whole new populations of students, and provides ASSET a safe haven for continuing its long-term mission to enhance STEM education and, ultimately, the STEM workforce.