Kent Golic - US grants

There are two main objectives for the proposed project period: first, techniques and vectors will be developed to extend the utility of the FLP site-specific recombinase as a tool for genetic manipulations; second, this technique will be applied to a mosaic analysis of the genetic factors controlling male fertility. Experiments that are aimed at elucidating how the three classes of male-sterilizing mutations differ in their modes of action will be performed. Of particular interest is the mechanism by which specific types of chromosomal rearrangement cause male sterility. The long term objective is to identify the genes that control and carry out the normal processes underlying this phenomenon, and characterize them in molecular detail. To these ends the following goals have been set: 1. A detailed analysis of the factors that influence the frequency of FLP-mediated recombination between FRTs will be performed. This will provide a solid footing for further work with this system. 2. Methods and vectors to perform site-specific integration of exogenous DNA will be developed. 3. Vectors and screening techniques will be developed to allow the easy recovery of site-specific chromosomal rearrangements. 4. Germline mosaics for three different classes of male-sterilizing mutations -- single gene mutations, Y chromosome fertility factors, and chromosomal rearrangements -- will be produced at different stages of spermatogenesis and compared. The experiments will help to define the mode of action by which each of these classes of mutation sterilizes males. The notion that spermatocytes function nonautonomously will be tested. 5. High-frequency dicentric formation will be employed to analyze the fate of dicentric chromosomes and the cells that carry them. The analysis will encompass cis-acting elements, the fate of dicentric- bearing cells, and the fates of dicentrics in the soma and in the germline. 6. A mutation screen will be performed to identify genes that function autonomously in spermatocytes. The project will provide insight into the genetic control of male fertility and into the cellular consequences of chromosome aberrations and aneuploidy.

DESCRIPTION: In spite of the remarkable success in the use of Drosophila as a model organism, a major disadvantage for workers in the field is the lack of a method of targeted gene disruption. Dr. Kent Golic proposes to develop two strategies for making gene knockouts that can be transmitted through the Drosophila germline. Both of these strategies take advantage of his expertise in utilizing FLP-mediated recombination in the fly. In the first of these strategies, the induction of FLP recombinase from a heat shock promoter will drive the excision as a circle of DNA sequences located between two FRT sites on a construct previously inserted into the genome. In some rare cases, this circle will then reintegrate into the genome by homologous recombination at sites where the genome contains target gene sequences related to those in the circle. In other cases, reintegration may be by "parahomologous targeting" in which chromosome rearrangements at the target gene accompany the integration event. The challenge then is to select for these rare reintegration events, which Dr. Golic intends to do with a novel strategy in which the excision of the circle will simultaneously inactivate a dominant male-sterile form of the beta2 tubulin gene and reconstruct a wild-type version of the same gene. As a result, male flies that were originally sterile should be able to make functional sperm in the descendants of germline stem cells where the excision occurred and where the wild-type beta2 tubulin gene was subsequently retained by virtue of its reintegration. Any fertile males are thus good candidates to have disruptions of the targeted gene; this will subsequently be verified by molecular techniques. In the second strategy, Dr. Golic will attempt to excise a linear fragment of DNA containing target gene sequences. The rationale is that the linear fragment should be more recombinogenic that circular DNA. Two different variants of this strategy will be tried, though both eventually will use the beta2 tubulin selection described above to identify animals in which the linear fragment recombined back into the genome. First, DNA located between two FRT sites will be excised as a circle by FLP recombinase as before. This circle will also contain a site for a rare-cutting endonuclease such as I-SceI. If expression of the nuclease is also under heat shock control, then the circle will be linearized. Alternatively, Dr. Golic will attempt to cut out the linear piece between two FRT sites by using FLP to make double strand breaks at the FRTs. This will require the identification of a mutant form of FLP that cuts at FRTs but does not recombine them. Dr. Golic describes a selection scheme in yeast to find such mutant forms of FLP, looking for altered FLPs that stimulate gene conversion at FRT by making double strand breaks.

Abstract for, "Mechanisms of Homologous Pairing in Drosophila" Kent Golic, P.I. - 9728070 The work described here is designed to characterize the mechanism of homologous chromosome pairing in mitotic cells of Drosophila rnelanogaster. Pairing will be examined in different tissues at different stages of development, in order to assemble a detailed picture of how cells manage to efficiently pair homologous sequences. The proposed experiments will determine whether pairing is a dynamic process that continues throughout interphase, and whether there are specific portions of the chromosomes that act to promote pairing. The influence of chromosome structure and the arrangement of chromosomes in the nucleus will be examined to determine whether these factors constrain the ability of chromosomal sequences to find and pair with homologous sequences elsewhere in the genome. In these experiments, the FLP site-specific recombinase and its target sites (FRTs) will be used to quantitate mitotic pairing, and the disruption of that pairing by chromosome rearrangements. The principal that underhes these experiments is that, in order to recombine, FRTs must pair, and that pairing must be brought about by normal cellular functions. By supplying the FLP recombinase to the cell in a controlled fashion, the levels of recombination that are detected must reflect the relative pairing of FRTs in different circumstances. Because FLP-mediated recombination is highly efficient, large amounts of data can be obtained with relative ease. In addition, FRT insertions can be used to assay pairing at many different sites throughout the genome. Chromosome pairing is a fundamental feature of chromosome behavior in many types of cells. Homologous chromosome pairing is needed for meiotic recombination and the faithful segregation of homologs in Meiosis I. Double-strand breaks can be repaired by using homologous sequences as a template, and this clearly requires that the sequences be in close proximity. Ectopic pairing and re combination has been implicated in small scale and large scale genome rearrangemerit in evolution. Pairing of sequences at ectopic sites may also be involved in generating the recurrent chromosome rearrangements that are associated with some types of cancer. Ectopic pairing may also underlie phenomena of multi-copy suppression that have been observed in many organisms. The work to be undertaken will increase our understanding of the mechanisms that cells use to pair homologs. It will likely be possible to generalize the results to provide a better understanding of the mechanism that cells employ to bring together homologous DNA sequences. In addition, the proposed work will have practical benefits for the Drosophila community. A resource of approximately 100 insertions of specially designed FRT-bearing P elements, with locations and orientations determined, will be produced. These P elements can be used for making chromosome rearrangements, as targets for the FLP-mediated insertion of genes, and for mitotic recombination. Furthermore, methods to increase the efficiency of FLP-mediated DNA integration should result.

DESCRIPTION: The mechanisms by which homologous chromosomes pair with each other remains mysterious; evidence can be mustered to support the existence or non-existence of discrete sites of pairing initiation. Historically, most interest in chromosome pairing has focused on the first meiotic division, where pairing is essential for recombination and proper chromosome segregation. Dr. Golic's lab has recently obtained a body of evidence supporting the idea that homologous chromosome pairing during mitosis serves as a close model for meiotic chromosome pairing. Most convincingly, he has in collaboration with Dr. Sergio Pimpinelli found genetic and cytological data that mitotic recombination during G2 causes homologous chromosomes to associate as a bivalent as they attach to the mitotic spindle. The basic thrust of this revised proposal is to use FLP-mediated DNA mobilization - a technique pioneered in the Golic lab - as a test system to explore the nature of homologous chromosome pairing in mitotic cells. In this approach, DNA sequences flanked by FRT sites are excised from one chromosomal location (the donor) by the action of FLP recombinase. The resulting FRT-bearing circles can reintegrate into a second chromosomal location (the target or recipient) that also contains an FRT site. In the first specific aim, Dr. Golic asks whether the original location of the donor in the genome influences the frequency of targeting. If the donor and recipient sites are far apart, will this reduce the frequency of mobilization? If this is not the case, it will imply that excised circles can diffuse freely through the nucleus. To answer this question, Dr. Golic will conduct the mobilization experiment using a variety of donor and recipient sites variably positioned on the same or different chromosomes. These experiments will utilize a sensitive somatic assay in which integration at a target site reconstructs a white+ gene, resulting in red clones in the background of a white-colored eye. The second specific aim is designed to extend previous observations that the larger the stretch of homology between the donor and target sites (outside of the FRTs they share), the more efficient the mobilization. This presumably results because homologous pairing helps bring the donor circle and target into proximity. Dr. Golic has devised a competition experiment in which cells will have two target sites with different amounts of homology with the donor. DNA from the non-preferred target site will then be added to the donor, to see if this alters target site bias. Control experiments will be performed to insure that any alteration in target site bias is due to the specific DNA sequences from the target site, rather than the length of the DNA segment employed. In the third specific aim, Dr. Golic will test whether certain candidate DNA sequences thought to be involved in pairing indeed play that role in the mobilization assay. These candidates include the PRE sequences recognized by Polycomb Group proteins, polymers of the Zeste protein binding site, repeats of the AG dinucleotide recognized by the GAGA protein (the product of the Trithorax-like gene), and other repetitive sequences. Candidate sites will be added to both donors and recipients to check for heightened rates of mobilization. Some of these experiments will be conducted as competition assays to see if candidate-containing donors will preferentially select targets with or without the candidate pairing sequence. The fourth specific aim will aim to adapt for Drosophila the "cassette exchange" method of FLP-mediated DNA integration developed by Schlake and Bode. This method employs two different FRT sites that cannot recombine with each other, so that integration of a sequences from a circular DNA molecule can occur only when two recombination events take place between FRT1 and FRT2 elements on both the donor and recipient. This technique should improve the efficiency of placing DNA at the target site because (in contrast to the current method), the integrated DNA cannot be lost subsequently by re-excision. The fifth specific aim details a clever experimental strategy which will exploit the findings of the four previous specific aims to identify DNA sequences that promote pairing with chromosomal target sites. The basis of this strategy is the injection of a library of random chromosomal fragments, cloned into an FRT cassette exchange vector, into embryos with an appropriate target construct containing a different antibiotic resistance gene. The FLP recombinase will be supplied in the form of a synthetic mRNA coinjected with the plasmid library. Plasmids containing sequences promoting plasmid/chromosome pairing should be favored for integration; these sequences should be recovered because the cassette exchange will switch the antibiotic resistance gene on the plasmid. Several rounds of this procedure should allow a high degree of enrichment of favored pairing sequences. The genomic location of DNA sequences recovered by this strategy will be determined to see if they contain a prominent pairing site or contain DNA sequences from the vicinity of the target element. The sixth and final specific aim will look at two pairing-dependent phenomena. The first of these is a variegated eye phenotype called pugilist-D, which is caused by a gene in which coding sequences for a biosynthetic enzyme are fused in frame to 1 kb of repeats of the GAGA factor binding site. Dr. Golic wishes to determine whether this pug-D phenotype results from the pairing of the fused gene back to centric heterochromatin containing the same repeated sequence. This will be done by using FLP-mediated DNA mobilization to place a pug-D transgene at different distances from centric heterochromatin. The second phenomenon explored in the last specific aim concerns the ability of a PRE sequence to silence an adjacent reporter gene in a pairing-dependent fashion. The question here is whether the silencing apparatus is reset every mitotic cycle. To this end, a construct will be made in which the PRE can be removed from its location adjacent to a white+ reporter by inducing FLP recombinase. If PRE function is indeed reset during mitosis, then the anterior region of the eye in front of the mitotic wave should show the normal variegation of the insertion (because this area is reset after the mitotic wave has passed over these cells), while the posterior region of the eye behind the mitotic wave should show strong reporter activity because silencing cannot be maintained in the absence of the PRE.

DESCRIPTION (provided by applicant): The ultimate goal of a genome project must be to understand what the genes are doing, not merely identify their existence. We are yet some distance from achieving that goal in Drosophila. The availability of a gene targeting method is but the first step towards that goal. Ultimately, it would be desirable to have a knockout mutation for every gene in the fly to be able to elucidate the function of each of those 13,600 genes. The targeting method that has been developed is vastly more efficient and effective than prior methods that have been developed for flies, but the method is in its infancy. Many parameters of the system are uncharacterized, and it is currently a lengthy procedure, requiring approximately 4-6 months from design of the targeting construct to the production of a knockout mutation. The proposed work is designed to increase our knowledge of the parameters that affect the efficiency of the method, leading to more efficient construct design and manipulation of the flies. A variety of methods to make gene knockouts will be tested and characterized to find those that are efficient and generally suitable for introducing mutations into specific genes. Experiments will also be undertaken to develop methods to speed up the process of making mutations, by weeks or perhaps months. The result of this work will provide a guide to the most efficient route towards producing specific alterations in the Drosophila genome. Moreover, the results will determine whether it becomes feasible to contemplate a large-scale knockout project in Drosophila, and what the time scale of such a project will be. Finally, the availability of efficient methods for gene targeting in Drosophila will aid the use of Drosophila as a model for understanding the biology of human diseases.

Genome sequencing projects are providing a wealth of knowledge about the genes carried in plant genomes. The ultimate goal of these projects is to understand the function of each of these genes. However, DNA sequence information typically only hints at potential function, and laboratory experiments are needed to characterize the details of gene action. The most powerful approach to characterizing gene function involves the analysis of mutant organisms. Thus, there is a pressing need for the development of methods that can use DNA sequence information to make directed modifications of plant genomes. The most desirable technique would give the ability to make a variety of directed modifications, and not be limited to functional knockouts. One such technique is gene targeting via homologous recombination, which is widely used in yeast and the mouse. However, a widely usable method for targeted modification of higher plant genes is not yet available.

The goal of this project is to produce a gene targeting methodology based on homologous recombination for higher plants. In this procedure, three constructs are introduced into the genome. The first and second are chimeric gene constructs that enable regulated expression of the Cre Recombinase and I-SceI genes. The third construct is a modified target gene placed between two loxP sites. The modified target gene contains an I-SceI recognition sequence inserted into the middle of the gene. In plants harboring all three constructs, expression of the Cre Recombinase and I-SceI genes is induced, these enzymes catalyze the formation of a broken-ended extrachromosomal DNA molecule in the nucleus, and homologous recombination between the extrachromosomal DNA molecule and the target gene generates two tandem copies of the target gene. This gene targeting procedure can be used to inactivate a target gene or to introduce modifications into a target gene.

To expedite development of a basic gene targeting procedure, Arabidopsis will initially be used as the test system. Its short generation time and small size will allow rapid technology development and testing. Furthermore, its small genome may increase homologous recombination frequency. The method to be used in these experiments provides the ability to produce an almost unlimited variety of genetic changes. Because the enzymes that carry out homologous recombination have been highly conserved throughout evolution, the methods we develop in Arabidopsis are very likely to work in a broad variety of species including commercially important crop plants. Thus, the methodology developed by the proposed experiments should benefit future efforts to improve crop yield and quality.

DESCRIPTION (provided by applicant): The long-term goal of this work is to use the model organism, Drosophila melanogaster, to investigate the genetic control of genomic imprinting. Genomic imprinting has been shown to be a general property of the Drosophila Y chromosome. Typically, a paternal imprint leads to repression of Y-linked reporter genes, while a maternal imprint gives relatively high expression of those genes. DNA methylation, histone methylation, histone acetylation, and chromatin structure are all candidates for generating a parent-specific imprint. We will investigate candidate genes that are known to be involved in these processes for their role in generating an imprint. We will also undertake a genome wide screen for genes that play a role in imprinting with a screen for quantitative or qualitative modifiers of the imprint. We will investigate reporter transgenes located in X chromosome heterochromatin for response to an imprint to determine whether imprinting is a Y chromosome phenomenon or a sex chromosome phenomenon. Finally, we will carry out sets of experiments aimed at elucidating the biological rationale for imprinting in Drosophila. Specifically, three hypotheses will be tested: that paternal imprinting is needed for proper expression of the Y fertility factors; that imprinting is a mechanism for silencing the transposon load carried by the Y chromosome; and, that imprinting is a mechanism for controlling expression of the rDNA. The results of these experiments will have implications for understanding human genetic disorders arising out of imprinting, and for the evolution of imprinting in general.

DESCRIPTION (provided by applicant): The loss of telomeric DNA in human somatic cells, arising from of the lack of telomerase expression, causes cellular senescesce and is associated with aging. Escaping the normal consequences of telomere loss is a critical step in the progression of cancer. Thus, understanding the normal mechanisms of cellular response to telomere loss and mechanisms that bypass the normal response are important for understanding, and possibly treating, cancer and ailments associated with aging. This work will use the model organism Drosophila melanogaster to investigate these issues. Preliminary experiments show that the response of Drosophila cells to telomere loss is very similar of the response of human cells to telomere loss, and is likely to be highly informative. There are 4 primary goals of this work. The first goal is to quantitatively characterize the response of Drosophila somatic cells to loss of a single telomere. A method for tracking the fate of cells that have lost a telomere will be implemented for this purpose. Second, the genetic control of these responses will be investigated. The third goal is to determine the mechanism of response of male germline cells, in which the non-telomeric chromosome ends are efficiently healed. The final goal is to investigate the nature of the healed chromosomes, to determine whether the telomeres they posses provide a normal protective function to the end of the chromosomes, or whether they are only partially functional. These investigations will provide an understanding of the machinery used to recognize telomere loss and to direct the response of cells to such loss. The differences between somatic and germline cells may provide insight into similar differences found in human cells.

? DESCRIPTION (provided by applicant): The long-term objective of this work is to understand the mechanisms that organisms use to ensure that genome integrity is maintained from generation to generation and from cell to cell. Genomes are subject to damage in a variety of forms and have evolved a variety of mechanisms to repair damage, or to cope with failure. To maintain chromosome stability, the linear chromosomes of eukaryotes must be capped with telomeres. Loss of a single telomere is a particularly challenging form of genome damage, and most often results in apoptosis, rather than successful repair. Two forms of repair are known for this type of damage: healing, which refers to the addition of a new telomere on the non-telomeric end of a chromosome; or Break-Induced Replication (BIR), in which the broken chromosome copies information from another chromosome to replace the end that it is missing. Both types of repair are associated with alterations in the genome. Since this is generally undesirable, these modes of repair may be considered repair of last resort. The work proposed here will investigate the germline responses to telomere loss. The goals are to identify the genetic controls that are used to detect and eliminate cells with this form of damage. Differences in the responses between male and female germlines will be explored. The experiments proposed here will also investigate the BIR mode of repair, which has not been previously demonstrated in any germline. This type of repair has additional interest because of its similarity to a form of telomere maintenance found in some cancer cells.

Project Summary This proposal describes investigations in three main areas to understand how the organization of chromosomes in the nucleus, and their differentiation into heterochromatin and euchromatin, impacts chromosome breakage and repair, chromosome pairing, and chromosome replication in Drosophila melanogaster. In the first part, dicentric chromosomes are generated in the male germline, where they typically break, delivering a chromosome with a broken end to each daughter cell. The influence of structurally distinct centromeres and the amount of centromeric histone CenpA on the fate of these chromosomes will be examined. Experiments will be undertaken to understand how cells with a broken chromosome choose their fate, to repair or to die, and how they choose between different modes of repair. These choices have obvious relevance for human health. When a cell lives, but fails to repair damage, it may become cancerous. The mode of repair can determine whether gametes transmit a normal genome, or a genome with deficiencies or other structural variants or mutations. One particular mode of repair, Break Induced Replication, is known to be mutagenic in yeast, and has been implicated in chromosome change in humans. Chromosomes repaired by BIR will be examined to determine the types and frequencies of mutations produced in the germline of a higher eukaryote. In the second part of this work, the mechanism of mitotic chromosome pairing will be examined. Significant progress has been made in identifying genes that promote or inhibit pairing, but how homologous regions of chromosomes find each other to initiate pairing is still a mystery. This work will test various models for the initiation of pairing by studying the frequency of site-specific recombination within and between rearranged chromosomes. This work also has human health relevance, since failures of meiotic pairing can lead to gametes with chromosomal aneuploidy. Some cancer cells also show inappropriate homologous pairing, implying a possible connection between these chromosome and cellular states. In the third part of the proposed work, the timing of DNA replication in heterochromatin will be examined. Mutations in genes that encode proteins that affect heterochromatin will be tested to determine their effects on replication timing. Proper maintenance of heterochromatin is important for genome stability, and disruption of its normal pattern of replication can lead to altered gene expression, with impacts on cellular function and health.

PROJECT SUMMARY/ABSTRACT The proposed Genetics Training Program (UGTP) at the University of Utah (UofU) will provide in-depth training in Genetics for a diverse group of 12 predoctoral students, arming them with scientific and professional knowledge and skills needed to conduct independent and rigorous research and become leaders within the biomedical research workforce. The U of U has a well-established, successful graduate training effort in genetics research, including a long-running Genetics T32 (45 years). The proposed new UGTP is an intensive two-year program of training in quantitative analysis, rigorous and transparent experimental practices, professional skills, the ethical practice of science, mentorship, and equity/inclusivity. These foundational objectives support the development of future multi-dimensional leaders and mentors in genetics research. The UGTP engages trainees at a formative stage of their career, in their 2nd and 3rd years of graduate school. Benefits of the program extend both before and after the intensive two-year training period. Completion of a pre-requisite first year graduate curriculum covering basic transmission genetics, ethical practices in research, critical thinking, hypothesis development, and understanding of rigor and reproducibility is required for the UGTP. Students who successfully pass a Capstone Exam administered at the end of Year 1, assessing trainee potential for independent research and rigor in scientific analysis and experimental thinking, are eligible for the UGTP. Each year about 12% of the approximately 50 TGE students are selected to become trainees. Trainees convene year-round in a suite of enriching activities. The central feature of the Training Program is a seminar course led by the PDs, limited to trainees, in which the students i) present research, critically evaluate progress, and discuss scientific best practices; undertake continued training in ii) RCR and iii) rigor, transparency, and reproducibility; participate in workshops on iv) professional development, v) mentoring; and vi) recognizing and responding to biased behaviors. Trainees present their research progress annually before the entire UofU Genetics community and select and host visiting genetics speakers throughout the year. Three advanced courses in quantitative and genetics analyses provide a breadth of training in the field of Genetics. The UGTP provides continuous mentorship in research and professional development: trainees develop IDPs with the Program Directors, adopt an extra mentor from the UGTP Steering Committee who provides guidance and communication with the UGTP, and participate in events aimed at career development and building an inclusive community. Following two years of UGTP-supported research with one of 61 UGTP faculty, trainees remain engaged through a novel initiative of near-peer mentorship, a program feature that supports the scientific and cultural development and mentoring skills of our trainees. The UGTP engages the entire UofU genetics community year-round through external speakers, Concepts in Genetics Seminars, and an Annual Retreat featuring trainee talks, plenary speakers, and poster presentations from the UofU genetics community.