To understand allele-specific regulation of gene expression, the maize pericarp color1 (p1) gene and its red pigmentation phenotype have been used as a genetic marker. Previous studies demonstrated that allelic variation at the p1 locus is a function of its gene structure, organization, and epigenetic regulatory mechanisms. Maize plants carrying the P1-wr (white pericarp & red cob glumes) allele produce ears with colorless kernel pericarp and red cob glumes and this phenotype is stable over generations. However, in the presence of Ufo1 (Unstable factor for orange1), the P1-wr allele confers variably enhanced pigmentation phenotypes in several tissues that include pericarp, cob glume, leaf sheath and husk. Ufo1 is a dominant modifier of p1 expression and it is unlinked from p1. Expression studies demonstrated that Ufo1-induced gain of p1 regulated pigmentation is associated with increased steady state levels of p1 RNA and decreased DNA methylation of P1-wr. To identify the nature of ufo1 gene and the mechanism of its action, further molecular and genetic characterization will be performed during this project. The first set of experiments will fine map the position of Ufo1 on chromosome 10S and these results will identify candidates for the ufo1 gene. The second objective will compare how the DNA methylation of single copy and multicopy alleles of p1 are affected by Ufo1. The third objective will examine the role of ufo1 in the maintenance of paramutation in two maize loci. This research will advance the current understanding of how overlapping epigenetic pathways uniquely regulate allele-specific patterns. The significance of this project is its elucidation of a class of modifiers of gene expression that can regulate or disrupt a well-programmed expression profile via transient changes in the DNA methylation or chromatin of specific target loci.
Eukaryotic gene regulatory mechanisms can restrict the expression of a gene to a specific tissue or a cell type. Most such patterns of expression are stably maintained due to the faithful transmission of alleles. Nevertheless, there are mechanisms that allow diversification of form and function. One such mechanism is epigenetic regulation operating through alterations in DNA methylation or in the packaging of DNA into chromatin. In this project the investigator will use the maize pigmentation regulatory gene p1 as a model system through which to understand the molecular basis of tissue specificity imposed in the form of alleles. to Understanding the basis of this unique type of allelic variability that exists in the form of natural and stable alleles is of great interest to biologists and plant breeders working to develop improved crops. This project will provide training for graduate and undergraduate students in the field of plant epigenetics. Furthermore, each summer the PI hosts two interns from the Pennsylvania Governor's School for Agricultural Sciences for independent study projects in maize genetics and molecular biology. The PI also participates in several mentoring programs for underrepresented undergraduates and collaborates with Dr. Sarwan Dhir of Fort Valley State University, a minority-serving institution in Georgia, in an NSF-funded REU program providing education and training to minorities and women in the field of plant biology, genetics and biotechnology.
Intellectual merit: Epigenetics is a science of genetics that does not follow laws of Mendel. In Mendelian or classical genetics if a gene is functional then it has its gene expression (function) turned 'ON' and we call that a ‘dominant allele’ of that gene but when a gene is mutated or in other words its DNA sequence is physically modified or gene is deleted etc then the gene does not function and is called a ‘recessive allele’. However, when the DNA sequence of a gene remains intact (i.e. no change in the DNA sequence) but certain chemical modifications on the DNA sequence makes the gene to become silent (or non-functional) then that gene is said to be epigenetically regulated (as oppose to genetically regulated in classical genetics). Two of the commonly known epigenetic causes are DNA methylation and chromatin modification. Both DNA methylation and chromatin modifications allow a DNA sequence to modify its conformation such that the new conformation does not allow that piece of DNA or gene to be functional anymore and thus the gene is called ‘silent’. What is most interesting is that these epigenetic modifications can become permanent marks and thus are inherited from one generation to the next. When that happens it is called transgenerational epigenetic inheritance such that he epigenetically influenced trait now can be transmitted over generations. Project outcomes: This research project aims to understand the epigenetic basis of gene expression modifications that take place in plant genes. In one of the aspects, we are interested in analyzing genetic modifiers (or the master proteins) that play a role in epigenetic modifications of gene expression of target plant genes. We ask an interesting and fundamental question that why there are multiple alleles at a single locus. We are using a model system in maize that has a conspicuous red pigmentation phenotype in maize kernels and cob glumes and this phenotype is controlled by maize pericarp color1 (p1) locus. The p1 locus has several different alleles that can be distinguished easily because of the functionality of p1 gene in kernel pericarp and cob glumes. Using the maize p1 system as a visual and genetic marker, we set out to identify the genetic modifiers that affect multiple processes in plants. We have previously characterized few alleles and found that these alleles differ with respect to their gene structure as well as their DNA methylation. Now we have found a genetic modifier called ufo1 (unstable factor for orange1) that can alter the expression of p1 alleles to produce a new alleles that are heritable over generations. The goals of this project were to fine map the ufo1 gene in the maize genome. By fine mapping we will know where exactly this gene is located on the chromosome. Our results show that the gene is located on short arm of chromosome 10. We then developed markers that are linked to this position of the chromosome. Using these markers now we will try to further come closer to the gene. Maize genome has been sequenced and we are using this sequence information to further develop new markers to continue fine mapping of the ufo1 gene. The second aspect of the project was to characterize the gene expression and DNA methylation of different p1 alleles that are affected by the ufo1 modifier. We have successfully performed these experiments which show that ufo1 gene is required to maintain silencing at these alleles of p1 by keep DNA methylation intact. We have also discovered that a dominant mutation referred here as Ufo1, has poor penetrance such that only a subset of F1 plants (cross: P1-wr; + x p1-ww; Ufo1) show the effect of Ufo1 mutation. In addition, we have discovered that Ufo1 plants have several growth and development defects and these types of abnormalities are expected when several different genes and transposons are modified for the DNA methylation within a plant’s genome. The study of gene expression stability (and instability) allows us to understand how different plant traits are inherited and how plants cope with different environmental stresses. Environment has a big influence on plant gene expression modifications and environmental influences are manifested via epigenetic changes that are then transmitted over multiple generations. Broader impacts: During this research project, two Ph.D. students, 11 undergraduate students and two postdoctoral fellows were mentored. The project also provided summer internships to four undergraduate students via the REU (Research Experience for Undergraduates) program of the NSF. Three peer reviewed papers were published in scientific journal, and students, postdoctoral researchers and the principal investigator presented their experiements via conference presentations.
