Progress 07/01/05 to 06/30/08
Outputs
OUTPUTS: Activities included cloning the required to maintain repression1 gene and establishing an experimental pipeline for position-based cloning of the remaining rmr genes. Experiments were conducted to characterize RMR1 function at the Pl1-Rhoades allele specifically and throughout the maize genome. These activities were synergistic with graduate student training for Jennifer Stonaker and Christopher Hale as well as undergraduate research mentoring for Agnes Choi and Negar Yaghooti. These activites were reported to the scientific community at the 48th, 49th, and 50th Annual Maize Genetics Conference, the 2007 Epigenetics Gordon Conference, the 2008 Keystone Symposium on Molecular Basis for Chromatin Modification and Epigenetic Phenomena and to the general public through publications in PLoS Biology, Science, and PLoS Genetics (2). Fundamental knowledge was obtained regarding the genetic silencing functions of RMR1 that highlight a novel areas of research for eukaryotic gene control and cereal genome function. Experimental results suggest that manipulation of RMR1 function can be applied to plant improvement programs. An invention disclosure was filed with the UC Berkeley Office of Technology and Licensing (Applications of the Required to Maintain Repression1 Gene Sequence in Plant Breeding; Case B07-96)and reported to iedison. A collaboration was initiated with Jose Gutierrez-Marcos to investigate the role of RMR1 in specific examples of transgene silencing. Improved germplasm resources related to US Patent #07264970 were developed and will be disseminated to the public through deposition to the USDA-ARS supported Maize Genetics Cooperation Stock Center. The project helped fund the thesis research and dissertation preparation for two UCB graduate students; Susan Parkinson, Ph.D and Stephen Gross, Ph.D. The project also help fund the research of three other UCB graduate students; Jennifer Stonaker, Christopher Hale, and Katherine McHenry. PARTICIPANTS: Jay Hollick (PD): Coordination, supervision, compliance issues, genetic experiments, training, teaching; Stephan Gross (UCB Graduate student): Bioinformatics and genetics training, professional development; Susan Parkinson (UCB Graduate student): Genetics training, professional development; Christopher Hale (UCB Graduate student): Genetics and molecular genetics training; Jennifer Stonaker (UCB Graduate student): Bioinformatics and molecular genetics training; Katherine McHenry (UCB Graduate student): Classical and molecular genetics training; Jana Lim (UCB undergraduate; lab assistant): Lab and nursery support; Negar Yaghooti (UCB undergraduate; Research Apprentice): Genetics and molecular genetics training; Agnes Choi (UCB undergraduate; Research Apprentice): Molecular biology training; Eric Chavez (UCB undergraduate; lab assistant): Nursery support; John Tran (UCB undergraduate; lab assistant): Nursery support; Jose Gutierrez-Marcos (University of Warwick, UK; collaborator): Transgene silencing collaboration, exchange of germplasm and preliminary research results. TARGET AUDIENCES: The academic audience broadly interested in eukaryotic cellular and nuclear mechanisms affecting gene and genome function was reached through dissemination of research results at two international meetings and through publication in the journal PLoS Biology. The industrial audience interested in mechanisms of gene silencing and heterosis were reached through dissemination of results at maize genetics meetings, through the issuance of a US patent, and through a University of California invention disclosure. PROJECT MODIFICATIONS: The original proposal was aimed at cloning the rmr2 gene. The genetic mapping approach became problematic as we encountered a region of low molecular polymorphism nearby the gene. Concurrent approaches to mapping the rmr1 gene were, however, successful in aiding it's molecular identification as encoding a novel plant-specific Snf2 protein. This is not considered a significant deviation from the original goals as the identification of rmr1 facilitates the same type of molecular insight into the molecular mechanism of paramutation.
Impacts
Agronomic traits that are based on transgenic technologies are often unstable due to poorly understood cellular processes that act to silence the action of transgenes within individual plants or entire populations. Known as "gene silencing," such uncontrolled instability negatively impacts gmo applications on both economic and social levels. Our project has identified the DNA sequences for a maize gene required for gene silencing which, together with existing technologies, now facilitates straight-forward strategies to mitigate, eliminate, or enhance gene silencing in maize and other crop species. Our discovery has the realistic potential to make gmo applications for plant improvement more cost effective, reliable, and to some degree, more socially responsible. We discovered this gene through our project to understand the genetic mechanism of paramutation, a process that generates and maintains epigenetic regulatory variation at specific Zea mays alleles. Using a pigment-based assay, this continuing project applied a half-saturation mutational screen to identify 10 unique loci encoding trans-acting factors required to maintain epigenetic repression of the purple plant1 allele, Pl1-Rhoades. To understand the molecular mechanism responsible for this process we cloned the rmr1 gene and discovered it encodes a SNF2-like protein distantly related to that encoded by Arabidopsis DEFECTIVE IN RNA-DEPENDENT DNA METHYLATION1 (DRD1). Together with recent identification of mediator of paramutation1 encoding a putative RNA-dependent RNA polymerase, the mechanism responsible for maintaining repressed paramutant states likely involves small RNA-dependent chromatin modifications. Molecular studies of Pl1-Rhoades are consistent with the RMR1/DRD1 protein assignment and further suggest a novel role for chromatin structure in affecting nascent RNA transcript stability. We also made the remarkable discovery that RMR1 is largely dispensable for maize genome homeostasis. Despite the absence of nearly 70 percent of the approx. 24nt RNA species, thought to be essential for "heterochromatin" maintenance, rmr1 mutant plant have been inbred for 11 generations without notable defects in plant morphogenesis or fertility. Our extension of the work allowed us to largely complete the cloning and initial characterization of the rmr1 gene as well as developing a pipeline for rapid position-based cloning of the remaining rmr genes. These research outcomes represent a fundamental change in our basic knowledge of cereal genome function and eukaryotic gene control. They highlight a fertile area for future research programs aimed at understanding the evolutionary and developmental roles of genetic mechanisms responsible for generating and maintaining epigenetic variation in higher plants.
Publications
- Hale CJ, Stonaker JL, Gross SM, Hollick JB. A novel Snf2 protein maintains trans-generational regulatory states established by paramutation in Zea mays. PloS Biology 5(10): 2156-2165 e275 (2007).
- Erhard, K.F., Stonaker, J.L., Parkinson, S.E., Lim, J., Hale, CJ., Hollick, JB. 2009 RNA polymerase IV functions in paramutation in Zea mays. Science 323: 1201-1205 doi:10.1126/science.1164508.
- Hale, C.J., Erhard K.F., Lisch D., Hollick, JB. 2009 Production and processing of siRNA precursor transcripts from the highly repetitive maize genome. PLoS Genetics 5(8): e1000598. doi:10.1371/journal.pgen.1000598.
- Stonaker, J.L., Lim, J.P., Erhard, K.F., Hollick, JB. 2009 Diversity of Pol IV functions is defined by mutations at the maize rmr7 locus. ("Accepted for publication" at PLoS Genetics)
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Progress 07/01/06 to 06/30/07
Outputs
OUTPUTS: Activities included cloning the required to maintain repression1 gene and establishing an experimental pipeline for position-based cloning of the remaining rmr genes. Experiments were conducted to characterize RMR1 function at the Pl1-Rhoades allele specifically and throughout the maize genome. These activities have been synergistic with graduate student training for Jennifer Stonaker and Christopher Hale as well as undergraduate research mentoring for Agnes Choi and Negar Yaghooti. These activites were reported to the scientific community at the 49th Annual Maize Genetics Conference, the 2007 Epigenetics Gordon Conference, and to the general public through publication in PLoS Biology. Fundamental knowledge was obtained regarding the genetic silencing functions of RMR1 that highlight a novel areas of research for eukaryotic gene control and cereal genome function. Experimental results suggest that manipulation of RMR1 function can be applied to plant improvement programs. An
invention disclosure was filed with the UC Berkeley Office of Technology and Licensing (Applications of the Required to Maintain Repression1 Gene Sequence in Plant Breeding; Case B07-96)and reported to iedison. A collaboration was initiated with Jose Gutierrez-Marcos to investigate the role of RMR1 in specific examples of transgene silencing. Improved germplasm resources related to US Patent #07264970 were developed and will be disseminated to the public through deposition to the USDA-ARS supported Maize Genetics Cooperation Stock Center. The project helped fund the thesis research and dissertation preparation for two UCB graduate students; Susan Parkinson, Ph.D and Stephen Gross, Ph.D.
PARTICIPANTS: Jay Hollick (PD): Coordination, supervision, compliance issues, genetic experiments, training, teaching; Stephan Gross (UCB Graduate student): Bioinformatics and genetics training, professional development; Susan Parkinson (UCB Graduate student): Genetics training, professional development; Christopher Hale (UCB Graduate student): Genetics and molecular genetics training; Jennifer Stonaker (UCB Graduate student): Bioinformatics and molecular genetics training; Jana Lim (UCB undergraduate; lab assistant): Lab and nursery support; Negar Yaghooti (UCB undergraduate; Research Apprentice): Genetics and molecular genetics training; Agnes Choi (UCB undergraduate; Research Apprentice): Molecular biology training; Eric Chavez (UCB undergraduate; lab assistant): Nursery support; John Tran (UCB undergraduate; lab assistant): Nursery support; Jose Gutierrez-Marcos (University of Oxford; collaborator): Transgene silencing collaboration, exchange of germplasm and preliminary research
results.
TARGET AUDIENCES: The academic audience broadly interested in eukaryotic cellular and nuclear mechanisms affecting gene and genome function was reached through dissemination of research results at two international meetings and through publication in the journal PLoS Biology. The industrial audience interested in mechanisms of gene silencing and heterosis were reached through dissemination of results at a maize genetics meeting, through the issuance of a US patent, and through a University of California invention disclosure.
PROJECT MODIFICATIONS: The original proposal was aimed at cloning the rmr2 gene. The genetic mapping approach became problematic as we encountered a region of low molecular polymorphism nearby the gene. Concurrent approaches to mapping the rmr1 gene were, however, successful in aiding it's molecular identification as encoding a novel plant-specific Snf2 protein. This is not considered a significant deviation from the original goals as the identification of rmr1 facilitates the same type of molecular insight into the molecular mechanism of paramutation.
Impacts
Agronomic traits that are based on transgenic technologies are often unstable due to poorly understood cellular processes that act to silence the action of transgenes within individual plants or entire populations. Known as "gene silencing," such uncontrolled instability negatively impacts gmo applications on both economic and social levels. Our project has identified the DNA sequences for a maize gene required for gene silencing which, together with existing technologies, now facilitates straight-forward strategies to mitigate, eliminate, or enhance gene silencing in maize and other crop species. Our discovery has the realistic potential to make gmo applications for plant improvement more cost effective, reliable, and to some degree, more socially responsible. We discovered this gene through our project to understand the genetic mechanism of paramutation, a process that generates and maintains epigenetic regulatory variation at specific Zea mays alleles. Using a
pigment-based assay, this continuing project applied a half-saturation mutational screen to identify 10 unique loci encoding trans-acting factors required to maintain epigenetic repression of the purple plant1 allele, Pl1-Rhoades. To understand the molecular mechanism responsible for this process we cloned the rmr1 gene and discovered it encodes a SNF2-like protein distantly related to that encoded by Arabidopsis DEFECTIVE IN RNA-DEPENDENT DNA METHYLATION1 (DRD1). Together with recent identification of mediator of paramutation1 encoding a putative RNA-dependent RNA polymerase, the mechanism responsible for maintaining repressed paramutant states likely involves small RNA-dependent chromatin modifications. Molecular studies of Pl1-Rhoades are consistent with the RMR1/DRD1 protein assignment and further suggest a novel role for chromatin structure in affecting nascent RNA transcript stability. We also made the remarkable discovery that RMR1 is largely dispensable for maize genome
homeostasis. Despite the absence of nearly 70 percent of the approx. 24nt RNA species, thought to be essential for "heterochromatin" maintenance, rmr1 mutant plant have been inbred for 11 generations without notable defects in plant morphogenesis or fertility. Our extension of the work is allowing us to largely complete the cloning and initial characterization of the rmr1 gene as well as developing a pipeline for rapid position-based cloning of the remaining rmr genes. These research outcomes represent a fundamental change in our basic knowledge of cereal genome function and eukaryotic gene control. They highlight a fertile area for future research programs aimed at understanding the evolutionary and developmental roles of genetic mechanisms responsible for generating and maintaining epigenetic variation in higher plants.
Publications
- Hale CJ, Stonaker JL, Gross SM, Hollick JB. A novel Snf2 protein maintains trans-generational regulatory states established by paramutation in Zea mays. PloS Biology 5(10): 2156-2165 e275 (2007).
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Progress 07/01/05 to 07/01/06
Outputs
The genetic process of paramutation generates and maintains epigenetic regulatory variation at specific Zea mays alleles. Using a pigment-based assay, this continuing project applied a half-saturation mutational screen to identify 10 unique loci encoding trans-acting factors required to maintain epigenetic repression of the purple plant1 allele Pl1-Rhoades. To understand the molecular mechanism responsible for this process, the primary objectives of this two-year project renewal were to clone the required to maintain repression2 (rmr2) gene and continue tool development for map-based cloning of three other rmr genes. Although several unforeseen obstacles prevent current identification of rmr2, the same approach led to the discovery that the rmr1 gene encodes a SNF2-like protein distantly related to that encoded by Arabidopsis DEFECTIVE IN RNA-DEPENDENT DNA METHYLATION1 (DRD1). Together with recent identification of mediator of paramutation1 encoding a putative
RNA-dependent RNA polymerase, the mechanism responsible for maintaining repressed paramutant states likely involves small RNA-dependent chromatin modifications. Preliminary molecular studies of Pl1-Rhoades are consistent with the RMR1/DRD1 protein assignment and further suggest a role for chromatin structure in affecting nascent RNA transcript stability. We are currently working to assign the Rmr1 5' gene structure in order to apply reverse genetic strategies and transgenic complementation to further validate rmr1 molecular cloning.
Impacts
Agronomic traits that are based on transgenic technologies are often unstable due to poorly understood cellular processes that act to silence the action of transgenes within individual plants or entire populations. Known as "gene silencing," such uncontrolled instability negatively impacts gmo applications on both economic and social levels. Our identification of the DNA sequences for a maize gene required for gene silencing, together with existing technologies, now facilitates straight forward strategies to mitigate, eliminate, or enhance gene silencing in maize and other crop species. Our discovery has the realistic potential to make gmo applications for plant improvement more cost effective, reliable, and to some degree, more socially responsible.
Publications
- No publications reported this period
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