Nancy E. Kleckner - US grants

DESCRIPTION (provided by applicant): The long-term objective of this project is to further elucidate the roles of mechanical forces for chromosomes and chromosomal reactions. Chromosome morphogenesis and dynamics will be examined from this perspective, comparatively for E.coli versus the prophase-to-prometaphase transition of eukaryotic chromosomes. Approaches will focus on high resolution 3D analysis, over time in the cell cycle, in wild-type and mutant situations, in living cells where ever possible. For E.coli, specific issues to be addressed will include the underlying physical and molecular basis for development of the nucleoid's helical ellipsoidal shape/density, the significance of this shape for dynamic chromatin movements, and the possibility that of cell division-related licensing of replication initiation. For eukaryotic chromosomes, the possibility of a meiosis-like intermediate will be explored. In complementary studies, our beads and bottlenecks magnetic micropiston will be used to probe the effects of compression and confinement on nucleoids/chromatin and DNA, including effects of real-time changes in buffer and molecular conditions. The roles of mechanical force for RecA- mediated homology recognition and strand exchange will be examined by parallel single molecule studies. Individual steps will be isolated and studied. The notion that reactions mediated by protein phosphatase PP2A are governed in important ways by the elasticity of its HEAT repeat scaffolding subunit will be further explored. Approaches to be used will include in vitro AFM analysis, in silico molecular dynamics, in vivo studies analysis of the PP2A-mediated response to spindle tension during the second division of meiosis, with budding yeast as an experimental system.

We will continue our isolation and characterization of mutations that affect transposase expression at steps subsequent to transcription initiation in order to determine why translation of the transposase gene is so rare, to assess the interplay between translation and messenger degradation, and to determine the roles of these events in IS10 anti-sense inhibition. We will analyze bacterium E.coli mutant strains lacking IHF and/or HU to determine the relative contributions of these, and any other related accessory host factors present in wild type cells, to the activity of IS10 outside ends and to host factor inhibition of transposase expression in vivo. We will also try to understand why E.coli uses IHF to regulate IS10 transposition (and other processes) by examining changes in IHF levels under different growth conditions. Finally, using strains lacking known host factors, we will isolate revertants in which outside end activity is restored in hopes of identifying genes for additional accessory proteins. Depending on the progress made in (1) and (2), we may initiate a new search for E.coli mutants defective in the transposition reaction.

The proposed experimentation addresses the structure and function of the synaptonemal complexes that form during meiosis in yeast. The proteins present in the complex will be isolated and characterized. Antibody directed against the proteins will be raised and then used to investigate the structure of the complexes using standard immunocytological techniques. The genes encoding the proteins will also be isolated. In related experiments, Dr. Kleckner will isolate and characterize both the rad50 gene and its product, and then determine the intracellular location of the product. Regulation of the rad50 gene, especially during meiosis will also be examined. Meiotic cell division, or meiosis, is an event common to the life cycles of virtually all eukaryotic organisms. During meiosis, homologous chromosomes pair and recombine, then segregate to the daughter nuclei. As yet, remarkably little is understood about the biochemical mechanism of eukaryotic recombination or its regulation. Paired chromosomes with sites of contact, or synapses have been observed in the microscope. The occurrence of such synaptonemal complexes appears to correlate with recombination. Some yeast mutants, in particular those affected in the rad50 gene, display lowered frequencies of recombination. The proposed isolation and characterization of the rad50 gene, its product and the protein components of the synaptonemal complex will lay a solid foundation for understanding the molecular events of eukaryotic recombination.

This award will support collaborative research between Prof. Nancy Kleckner of Harvard University and Dr. Olivier Huisman of the Institut Pasteur, Paris, in the area of genetic analysis of mutant proteins. The objective of this work is the structural and functional dissection of DNA sequences at the ends of a gene segment, which is capable of moving from place to place in the chromosomes of an organism. Such a gene segment is called a transposon; the particular transposon to be studied in this work is called Tn10. Transposition of transposons leads to mutation, and the mutations which occur are to be studied by both genetic and biochemical methods. In the latter, assays for the binding of important proteins to specific DNA sequences should reveal the effects of specific mutations on known processes, and could show new intermediates in the reaction which might accumulate due to intermediate mutational steps. The French collaborators have considerable experience in genetic analysis. Additional materials, as well as expertise in use of an in vitro biochemical system for analysis of the Tn10 transposition, will be brought to the project by the U.S. investigator. The knowledge of complex interactions between DNA and proteins that is expected to result from this research could lead to enhanced progress in our understanding of the processes of the generation, and therefore of alteration, of life itself.

The purpose of this workshop is to assemble for discussions a select but representative group of scientists who are actively working at the forefront of research on the biochemical mechanisms used in site-specific recombination and transposition. This interrelated group reactions lie at the heart of a number of very important biological processes: integration of retroviruses generation of antibody diversity, transmission of antibiotic resistance genes, rearrangement of bacterial chromosomes and mobility of eukaryotic introns. The oral presentations at this Workshop will emphasize the mechanistic aspects of these recombination reactions. There is currently rapid progress in this area, and a specialized meeting for leaders in this field should be both timely and productive. The roster of speakers includes several scientists who have pushed the analysis of recombination for the first time into the realm of enzyme kinetics, as well as many scientists who have applied other types of biochemical, genetic and topological analysis. Topics will include retroviral integration, reactions of the integrase and resolvase families, Holliday junctions, immunoglobulin rearrangements, prokaryotic transposable elements, and Drosophila P elements. We are also encouraging poster presentations from scientists working in systems which are not yet amenable to mechanistic analysis. This should serve both to broaden the biological base of the meeting and to encourage those working in relatively undeveloped systems to undertake a more mechanistic approach. The meeting will be limited to 100-135 participants. This number is sufficient to include all of the principal investigators who are working productively in this area as well as a reasonable sampling of post-doctoral fellows and an occasional outstanding graduate student. However, this number is small enough that efficient interchange amongst all the participants is still possible.

Partial support is requested for a Workshop on Site-specific Recombination and Transposition to be held on September 12-16, 1990, at the Marine Biological Laboratory in Woods Hole, Massachusetts. The purpose of this workshop is to assemble for discussions a select but representative group of scientists who are actively working at the forefront of research on the biochemical mechanisms for site-specific recombination and transposition. This interrelated group of reactions lie at the heart of a number of very important biological processes: integration of retroviruses including those responsible for AIDS and certain forms of cancer, generation of antibody diversity, transmission of antibiotic resistance genes, rearrangement of bacterial chromosomes, and mobility of eukaryotic introns. The oral presentations at this Workshop will emphasize the mechanistic aspects of these recombination reactions. A specialized meeting for leaders in this field should be both timely and productive. There has been rapid recent progress in many different systems. Furthermore, it is becoming increasingly apparent that reactions occurring in eukaryotic organisms are intimately related to those identified in bacteria: integration of AIDS virus and other retroviruses is mechanistically closely related to integration of bacterial transposons, site specific immunoglobulin rearrangements are closely related to prokaryotic site specific recombination reactions, etc. In promoting the exchange of insights and information from different systems, the Workshop should greatly enhance our basic understanding of all of these reactions. In addition, in the particular case of AIDS virus and other retroviruses, it should further the goal of using the recombination reaction as a target therapeutic intervention. The roster of speakers includes scientists who have applied biochemical, genetic and topological analysis including several who have pushed the analysis of recombination for the first time into the realm of enzyme kinetics. Topics will include retroviral integration, reactions of the integrase and resolvase families, Holliday junctions, immunoglobulin rearrangements, prokaryotic transposable elements, and Drosophila P elements. We are also encouraging poster presentations from scientists working in systems which are not yet amenable to mechanistic analysis. This should serve both to broaden the biological base of the meeting and to encourage those working in relatively undeveloped systems to undertake a more mechanistic approach. The meeting will be limited to 100-135 participants. This number is sufficient to include all of the principal investigators who are working productively in this area as well as a reasonable sampling of postdoctoral fellows and an occasional outstanding graduate student. However, this number is small enough that efficient interchange amongst all of the participants is still possible.

DESCRIPTION (provided by applicant): Meiosis is the specialized cellular program that produces haploid male and female gametes from a diploid parental cell as required for sexual reproduction. Our research addresses the central unique feature of this program, a complex series of interactions between maternal and paternal homologs, in three areas: (I) Recombination-independent pairing. This process, which also occurs in mitotically-dividing cells of some organisms ("somatic pairing"), is one of the major chromosomal behaviors for which there is no hint of the fundamental molecular mechanism. We will apply high-throughput 3C analysis to identify pairing "hot spots" and to examine the roles of specific variables of interest to pairing at a single defined locus. We will further analyze pairing interactions by motion correlation analysis of GFP-tagged loci in living cells. As time permits, we will begin to investigate the possibility of isolating (and then characterizing) paired molecules. (II) Recombination-related events. We will continue to analyze the basic nature of recombination at the DNA level by isolation and AFM visualization of recombination intermediates (notably double Holliday junctions) from a single recombination "hot spot". We will initiate a study of crossover interference by searching for functions which, when overproduced, increase or decrease the robustness of this process;also, we will try to distinguish among possible models for interference by creating, and assessing the effects of, a defined "loop" along a chromosome. Finally, we will continue to explore the roles of axis components for recombination by: studies of the "mitotic" cohesin Mcd1/Scc1/Rad21;high resolution analysis of local variations in protein localization via linked MNase studies;and further definition of chromosome organization via high resolution analysis of cohesin and condensin localization along pachytene chromosomes. (III) Motion and mechanics. We will improve our methodology for isolation of individual pachytene chromosomes as objects for mechanical studies. We will pursue our recent discovery of actin- and telomere-based chromosomal motion using existing reagents and by searching for "motion mutants". Defects in meiotic interhomolog interactions underlie many types of hereditary infertility as well as most chromosomal aneuploidies, all of which confer significant societal burdens (e.g. Downs syndrome). Basic understanding of these processes is crucial for diagnosis and potential amelioration of these conditions.

This award supports Professor Nancy Kleckner of Harvard College and her graduate student, Ruth Padmore, M.D., to collaborate in molecular biology research with Professor Christa Heyting and others of the Department of Genetics of the Agricultural University of Wageningen, The Netherlands. Both of these laboratories are interested in understanding the mechanisms of and relationships between chromosome pairing and recombination during meiosis. However the two laboratories have used very different approaches and very different organisms for their analysis, yeast and rats. The overall scientific goal of the collaboration is a comparative analysis of the processes of interest in the two organisms. The complementary skills of the two research groups in yeast molecular genetics and mammalian cell biology and cytogenetics should accelerate progress in both laboratories. Meiosis is essential for sexual reproduction. One of the most important aspects of meiosis is the pairing of and recombination between homologous chromosomes. The general goal of this collaboration is a functional comparison of these processes in yeast and higher eukaryotes. Most work on the genetics of meiosis has been done with yeast, while most work on the cytology of meiosis is done with mammals. It is important but difficult to relate the two lines of research.

The Harvard University Faculty of Arts and Sciences Training Grant in Genetics, directed by Professor Nancy Kleckner, currently supports 20 predoctoral students in the Department of Molecular and Cellular Biology (MCB) and Organismic and Evolutionary Biology (OEB). The 422,000 net sq.ft. training facilities are located in Cambridge, MA in the Fairchild Biochemistry Building, the Biological Laboratories, the Museum of Comparative Zoology and the Herbarium. Graduate programs in the participating Departments involve both course work and research and lead to the Ph.D. degree. These programs provide the promising young scientists who matriculate in these Departments an opportunity to learn and develop in an exciting and challenging scientific environment. The current positions of former trainees attest to the strength and success of the Genetics Training Program in previous years. The specific goal of the Genetics Training Program is to provide predoctoral students with a sophisticated and rigorous training in genetics that enables them to appreciate and to practice genetics as a primary experimental approach to important biological problems. Graduate students in the program are required to take an advanced course in genetics during their first year and to serve for at least one semester as a teaching fellow in a course with a strong genetic component, in addition to fulfilling other graduate training requirements. Students trained in the Program are also expected to integrate genetic approaches with approaches provided by other disciplines. The training faculty for the Genetics Program now numbers 23. Scientific areas of interest represented among these faculty include neurobiology, development, DNA and chromosome behavior, gene expression, cell motility, cellular organization and compartmentalization, sexual differentiation, population biology, ecology and evolution. The core of the training faculty comprises the 14 members of the Genetics Consortium, a newly self- appointed group charged with overseeing the discipline of genetics on the Cambridge campus of Harvard University. Faculty of the Genetics Consortium are responsible for most of the formal course work in genetics on this campus at both the graduate and undergraduate levels. The remaining 9 training faculty are members whose current work has a significant genetic component. All faculty of the Genetics Training Program participate as research supervisors for predoctoral trainees. The Genetics Training Program supported by this grant began in 1978. The Program has been drastically restructured within the past year, for the first time since its inception. A series of changes provides greater focus on genetics as a discipline, a strong didactic component in genetics and a number of initiatives that not only enhance the Program specifically but also keep genetics in a position of prominence within the local scientific community as a whole. Support is requested for 16 trainees in each of the next five years.

DESCRIPTION (provided by applicant): This application addresses the interactions between homologous chromosomes during meiosis in yeast. (I) DSB-independent pairing. We will use FISH to probe DSB-independent homolog pairing with respect to relative abundance in R- and G-bands, relationship to sister chromatid cohesion and involvement of chromatin structure proteins. We will use "Capturing Chromosome Conformation" (3C) methodology to identify sites of pairing contacts. We will carry out a pilot experiment to investigate a possible new assay for pairing-defective mutants. (II) Initiation of recombination: the DSB transition. We will investigate whether pre-DSB recombinosomes are physically associated with their underlying chromosome axes by 3C methodology and genetic studies. We will also use 3C methodology to identify nascent DSB/partner interactions and to explore homolog/sister discrimination. We will further explore constraints governing the number of DSBs that can occur at a single locus in any give meiotic nucleus. (III) Later stages of recombination. We will further explore meiosis in mutants that affect the crossover control transition, with attention to important effects of temperature. We will continue analysis of the bouquet stage in wild type and selected mutants. We will continue analysis of the role of Mlh3 for meiotic recombination. We will examine the phenotypes of mutations suspected to affect conversion of single-end invasions to double Holliday junctions at mid-pachytene. And we will continue to investigate which topological isomer(s) of double Holliday junctions occur during meiosis. (IV) Meiotic chromosome structure and mechanics. We will further explore chromatin/axis/sister interplay revealed by our recent studies. We will use 3C methodology to investigate physical properties of mid-prophase chromosomes. We will examine the dynamics of synaptonemal complex twisting in vivo. We will begin to develop methods for isolating, analyzing and physically manipulating pachytene chromosomes in vitro.

Abstract Chromosomes are the repositories of our genetic material. We consider them to be living, breathing objects whose fluctuations in time and space underlie their most basic functions. By comparing meiotic and mammalian mitotic chromosomes and E.coli nucleoids we seek to identify fundamental commonalities. Meiosis underlies sexual reproduction. Its unique hallmarks are pairing and recombination of maternal and paternal homologs, including the phenomenon of crossover interference in which crossover sites occur with even spacing along the chromosomes. We analyze this patterning process by 4D long timescale visualization in our new C.elegans platform and by cytogenetic studies in the fungus Sordaria. We are probing our new mechanical model and our discovered inter-homolog structure/DNA bridges, concomitantly analyzing new-found players and identifying more. For pairing, with our new low SNR spot detection algorithm and FROS tags in budding yeast, we probe partner searching and homology identification. In Sordaria, our first-ever comprehensive screen of meiotic long noncoding RNAs will identify species involved in patterning and/or pairing. Other studies investigate the evolution of meiosis from mitosis and evolution of stable autopolyploidy. Mitotic chromosomes start in a diffuse but spatially ordered state (G1), but ultimately evolve into compact, side-by-side sister chromatids ready for segregation. We are pursuing our discovery of inter-sister structure/DNA bridges and their emergence via axial torsional stress by quantitative modeling. Using live cell imaging of mammalian chromosomes, including our new 4D long timescale platform for fluorescent speckle microscopy, we are exploring our finding that metaphase chromosomes are folded, not coiled, and will ask when/how G1 chromosomes acquire their disposition, with/without our proposed compaction/expansion cycles. E.coli chromosomes also undergo global compaction/expansion cycles, as we discovered. Now, by high throughput 4D imaging of cells growing in agarose grooves, and of membrane-enclosed L-forms, we are investigating the (supercoiling-dependent) mechanism of these cycles; their roles for sister segregation and cell division; and the roles of nucleoid/membrane interactions in both aspects. We are also working to reconstitute nucleoid cycles in vitro, and are asking if cycles also occur in other bacteria. For many of the above studies, chromosomes can be viewed as mechanical objects, subject to deforming forces (stresses) that drive local and global movement, abrupt changes or, via stress redistribution, spatial patterning. To directly detect and analyze such effects, we are developing ZnS-Mn mechano- luminescent nanocrystals as a non-invasive in vivo stress sensor. Once developed, this tool will be applied to detection of waves and/or other, yet-to-be imagined, stress patterns in mammalian chromosomes. Our unique studies will provide novel entry points into problems of infertility and birth defects (meiosis), genetic instability and cancer (mitotic cells) and antibiotic resistance (E.coli L-forms).