Progress 07/01/01 to 06/30/04
Outputs
The goal of this multi-award project was to identify genetic components required for an unusual plant genetic mechanism, known as paramutation, causing heritable changes in gene regulation. A model system affecting pigment production was identified in maize operating on a specific allele of the purple plant 1 locus (pl1) and a half-saturation mutational screen was conducted to identify relevant genes encoding components required for paramutation. Nine distinct required to maintain repression (rmr) genes were identified by mutational analysis and breeding stocks were created to understand their specific functions at the pl1 locus and, in a broader context, their roles in genome-wide regulation. Potential commercial importance of these materials in relation to amelioration of transgene silencing was disclosed and is currently under review in the US Patent filing: 2001 09/972,805 Genetic Functions Required for Gene Silencing in Maize. Chandler, V.L., Hollick, J.B.,
Dorweiler, J.E., Lisch, D., Kubo, K. and C. Carey. Molecular expression studies of pl1 transcriptional regulation in rmr mutant plants led to a working model to explain the paramutation mechanism operating at pl1. Tests of this model highlight important differences with seemingly similar mechanisms in other eukaryotes. We used mutational analysis, genetic segregation, pl1 RNA measurements, and genetic mosaic analysis to test a post-transcriptional component of pl1 paramutation. Specific mutant pl1 alleles, selected for pigment defects, were found to be deficient in paramutational properties thus genetically defining specific DNA sequences involved in the process. These materials formed the basis of a project currently funded by NSF to functionally identify these DNA sequences. Mosaic analysis showed that paramutation is a progressive process occurring throughout plant development and also reveals the process is clonal in nature without systemic spread of the paramutation behavior.
Molecular and genetic tests further identified additional genomic targets of Rmr action including specific transposons families and developmental regulators thus providing additional experimental systems for future study. As further progess in understanding the paramutation mechanism requires identifying the molecular nature of rmr genes, we employed high-throughput technologies to identify rmr2 and rmr6 candidate genes. Based on highly refined genetic map position and subsequent comparisons with NSF-funded maize genomics resources, we identified a gene encoding a putative histone methyltransferase enzyme which could be involved in chromatin-level changes associated with the paramutation mechanism. Consistent with the behavior of these proteins, we found diagnostic patterns of cytosine methylation altered in rmr2 mutant plants. We also refined the map position of rmr6 and found organization of this region to be highly conserved with that of the sequenced rice genome. Based on these
comparisons, there are several likely candidate genes to consider. Thus we made significant strides towards identifying the molecular nature of the paramutation process through candidate identification of at least one rmr gene.
Impacts
Our research progress suggests that the genesis and maintenance of heritable epigenetic variation in plants can be mediated by RNA interactions that affect the heritable transcriptional regulatory status of affected genes. Although key aspects of this mechanism are still unknown, it is expected that a molecular understanding of this RNA-based mechanism will lead to improvements in transgene vector designs and molecular screening techniques for transgenic approaches to crop improvement. Similarly, novel approaches for targeting heritable epigenetic silencing of specific loci may be developed as tools for studying gene structure and function. Further, manipulation of RMR functions through transgenic or traditional breeding may allow reliable control of traits governed by genetic modifications.
Publications
- Hollick, J.B. (2003) Paramutation in plants, in Encyclopedia of Life Sciences, Macmillan Reference Ltd., London.
- Hollick, J.B. and Chandler, V.L. 2001 Genetic factors required to maintain repression of a paramutagenic maize pl1 allele. Genetics 157: 369-378.
- Dorweiler, J.D., Carey, C.C., Kubo, K.M., Hollick, J.B., Kermicle, J., Chandler, V.L. 2000 Mediator of paramutation 1 (mop1) is required for the establishment and maintenance of paramutation at multiple maize loci. The Plant Cell 12 (11): 2101-2118.
- Hollick, J.B., Patterson, G.P., Asmundsson, I.M., Chandler, V.L. 2000 Paramutation alters regulatory control of the maize pl locus. Genetics 154: 1827-1838.
- Hollick, J.B. and Chandler, V.L. 1998 Epigenetic allelic states of a transcriptional regulatory locus exhibit overdominant gene action. Genetics 150: 891-897.
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Progress 01/01/03 to 12/31/03
Outputs
Our working model for pl1 paramutation remains intact, yet results from the prior year have served to highlight important differences with seemingly similar mechanisms in other eukaryotic organisms. We used mutational analysis, genetic segregation, pl1 RNA measurements, and a genetic mosaic analysis to test a post-transcriptional component of pl1 paramutation. Specific mutant pl1 alleles, selected for pigment defects, were found to be deficient in paramutational properties thus genetically defining specific DNA sequences involved in the process. The fact that subsequent molecular analyses indicate pl1 RNA is still produced at low levels in these defective alleles modifies our current working hypothesis concerning the functional role of pl1 RNAs in this process. A completed genetic mosaic analysis shows that paramutation is a progressive process occurring throughout plant development. This analysis also reveals that the process is clonal in nature without any systemic
spread of the silencing behavior. We also employed high-throughput technologies to identify candidate genes for both rmr2 and rmr6. Based on a highly refined genetic map position and subsequent comparisons with NSF-funded maize genomics resources, we have focused our attention to a gene encoding a putative histone methyltransferase enzyme which could be involved in chromatin-level changes associated with the paramutation mechanism. DNA sequence analysis of our rmr2 mutant alleles will serve to confirm or potentially discount this candidate gene assignment. Consistent with the behavior of these proteins, we found diagnostic patterns of cytosine methylation altered in rmr2 mutant plants. Tests are now being formulated to functionally assess this assignment. We also refined the map position of rmr6 and found the organization of this region to be highly conserved with that of the sequenced rice genome. Based on these comparisons, there are several candidate genes currently under
consideration. Thus we have made significant strides towards identifying the molecular nature of the paramutation process through candidate identification of at least one rmr gene. Previous analyses indicating rmr-based effect on Mutator transposons motivated our experiments to test whether or not the movement of maize transposons, like Activator (Ac), were affected. We completed stock constructions and phenotypic analyses showing that transpositions of Ac from a reporter locus were unaffected by RMR functions. These data indicate that RMR functions are not generally involved in specifying transposon regulation per se. We also made progress in genetically mapping other rmr loci. We positioned rmr7 to the short arm of chromosome 2, rmr1 to the long arm of chromosome 9, and rmr11 to about a 20% region of the genome not covered by our series of BA translocation stocks. With these crude positions, we can now employ high-throughput mapping to help identify candidate genes.
Impacts
Our research progress suggests that the genesis and maintenance of heritable epigenetic variation in plants can be mediated by RNA interactions that affect the heritable transcriptional regulatory status of affected genes. Although key aspects of this mechanism are still unknown, it is expected that a molecular understanding of this RNA-based mechanism will lead to improvements in transgene vector designs and molecular screening techniques for transgenic approaches to crop improvement. Similarly, novel approaches for targeting heritable epigenetic silencing of specific loci may be developed as tools for studying gene structure and function. Further, manipulation of RMR functions through transgenic or traditional breeding may allow reliable control of traits governed by genetic modifications.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
We are investigating the regulation of color production in maize to understand chromosome-based gene control mechanisms in higher plants. Visual pigment levels are a simple indicator of quantitative gene expression from the purple plant 1 (pl1) locus. One particular form of the pl1 gene is subject to epigenetic switching from high expression states (lots of pigment) to low expression states (very little pigment). The low expression state is very stable and invariably causes a highly expressed partner to switch to the low expression form. This unusual type of gene silencing is known as "paramutation." During the initial phase of this project, we identified 8 novel genetic loci whose functions are required to maintain repression of the particular pl1 gene; they are responsible for maintaining the low expression state. We also had hints that RNA transcripts from the pl1 gene were needed to cause paramutation. This gene silencing mechanism appears to be of broad
significance to proper genome function. Mutations in various RMR components lead to molecular changes at transposable elements and cause developmental abnormalities. The goals of the current project are to investigate the potential role of an RNA-based mechanism in mediating paramutation, proceed with molecular cloning of the rmr2 and rmr6 genes, assess the role of RMR1, RMR2, and RMR6 functions on the regulation of various maize transposons, and to genetically map new rmr loci. We determined that pl1 RNAs, but not functional PL protein, are required to establish paramutation silencing. Mutant analyses indicate that both transcriptional and post-transcriptional mechanisms are need to effect heritable gene silencing. We are currently using molecular tests to decipher the difference between an inducing pl1 RNA versus a normal, non-inducing, pl1 RNA. Molecular cloning of the rmr6 gene is progressing by two strategies; transposon-based co-segregation analyses and position-based candidate
gene approach. We have two candidate genes identified from a map-based cloning strategy for rmr2. Stock constructions have been completed for the tests of RMR functions in the regulation of two maize transposons and results will be known by next month. As a prelude to eventual molecular cloning, we have mapped two new rmr loci using both cytogenetic tools and standard genetic linkage analyses. Excellent progress has been made on all specific aims of this project during the last year. Molecular identification of the rmr6 and rmr2 gene products is currently our top priority.
Impacts
Progress on this project suggests that the genesis and maintenance of heritable epigenetic variation in plants can be mediated by RNA interactions that affect the heritable transcriptional regulatory status of affected genes. Although key aspects of this mechanism are still unknown, it is expected that a molecular understanding of this RNA-based mechanism will lead to improvements in transgene vector designs and molecular screening techniques for transgenic approaches to crop improvement. Similarly, novel approaches for targeting heritable epigenetic silencing of specific loci may be developed as tools for studying gene structure and function.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
We are beginning to clarify the role of a post-transcriptional regulation in a gene-silencing mechanism operating at the Maize pl1 locus. Research completed during the initial phase of this project continues to support the hypothesized role of pl1 RNA as a mediator of trans-silencing interactions occurring at pl1. This evidence is derived from a combination of genetic and molecular expression experiments. Standard genetic crosses with specific cytogenetically-defined chromosomes demonstrate that certain derivative alleles of the pl1 locus displaying a "loss-of-function" phenotype fail to cause trans-silencing of specific pl1 alleles. Measurement of pl1 RNA using RNase-protection assays indicate that all derivative pl1 alleles of this type fail to produce full-length pl1 RNA transcripts, thus implicating a functional role for pl1 RNA in the trans-silencing mechanism. This evidence complements previous findings in which derivative pl1 alleles presumed to make
non-functional PL proteins still retain the ability to cause trans-silencing of specific pl1 alleles. We have made progress towards understanding the role of this gene-silencing mechanism in the control of Maize transposable elements by advancing stock constructions for specific genetic tests of the requirement for RMR functions for transposition behaviors. We have also made progress towards identifying the molecular nature of several genetically-defined components required for this type of gene-silencing. The genetic map positions of the rmr2, rmr6, and rmr7 loci have now been identified and refined using special cytogenetic mapping tools and ongoing genetic linkage studies. Candidate gene approaches for molecular cloning are routinely being evaluated as mapping information from NSF-funded projects continues to be deposited in the public domain. In a complementary approach, we are currently pursuing "transposon-tagging" methodologies using mutable alleles of rmr6 and rmr2. We have
generated segregating populations and collected tissues needed for the type of standard co-segregation analyses used to molecularly clone Maize genes.
Impacts
Initial progress on this project suggests that the genesis and maintenance of heritable epigenetic variation in plants can be mediated by RNA interactions that affect the heritable transcriptional regulatory status of affected genes. Although key aspects of this mechanism are still unknown, it is expected that a molecular understanding of this RNA-based mechanism will lead to improvements in transgene vector designs and molecular screening techniques for transgenic approaches to crop improvement. Similarly, novel approaches for targeting heritable epigenetic silencing of specific loci may be developed as tools for studying gene structure and function.
Publications
- No publications reported this period
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