Progress 02/15/11 to 02/14/16
Outputs
Target Audience:Plant scientists, rice breeders and resarchers, molecular biologists, biochemists, Changes/Problems:While examining the mutant phenotype of the genes encoding the chromatin remodeling complex proteins, we observed certain degree of phenotype variation over different generations. This could be due to the nature of epigenetic trait, which delayed the publication of our major results because extra verification experiments have to be performed. Although we are successful in purifying other chromatin modification protein complex such as the polycomb protein complex, we were not so lucky with SWl/SNF complex purification. It is possible that the complex is not very stable or the the epitope tag we used interfering with the complex stability. What opportunities for training and professional development has the project provided?Two graduate students, one postdoctoral research associate, and one undergraduate student received training by joining in this project. In addition, the PI has actively introduced chromatin structure, chromatin remodeling, and chromatin modifications to Molecular Biology, Biochemistry, and Plant Molecular Biology courses over the last five years. How have the results been disseminated to communities of interest?We have had seven peer reviewed publications, six oral presentations worldwide, four meeting posters, and two large data set deposits to existing database. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In eukaryotic cells, the DNA molecule is wrapped around histone proteins to form a structure called chromatin. The basic structural and functional unit of chromatin is nucleosome. Studies have shown that the positioning of nucleosomes along the DNA molecule profoundly influences gene expression and positioning of nucleosome is dynamically regulated. The proteins that regulate the nucleosome position usually act as complexes and these complexes are called chromatin remodeling complexes. In plants, the composition and structure of plant chromatin remodeling complexes and the molecular mechanisms underlying chromatin remodeling mediated gene regulation are still poorly explored. This project aims to study the function of SWI/SNF chromatin remodeling complex in the regulation of gene expression and plant development in rice. We isolated the mutants of all the genes encoding SWI/SNF proteins and examined the phenotypes of these mutants with emphasis on seed development and grain nutrition regulation. In addition, we examined the correlation between nucleosome position and gene expression. Our results demonstrated that chromatin remodeling genes play an essential role in gene expression and in multiple stages of rice plant development, including grain filling. Our results also indicate that nucleosome positioning may interact with some non-coding RNAs in plants, a result that has not been revealed in related chromatin remodeling studies in other organisms. Our research results advanced the understanding of the biological function of chromatin remodeling in plant development and gene regulation. We isolated 18 mutants of the 11 putative genes encoding subunits of the SWI/SNF complex and two histone deacetylase genes that encode proteins interacting with chromatin remodeling complex. While 14 of the mutations have homozygous mutants available, four of the mutations can only generate heterozygous plants, suggesting that mutations of these four genes may have lethal effect. Among the homozygous mutants, three of them displayed phenotypes in both vegetative and reproductive stages. The others had no visible phenotype. Probably due to the epigenetic nature, however, the phenotypes displayed some degree of variations over generations, which delayed our publication substantially. To further confirm the results, we have constructed multiple RNAi constructs to generate RNAi transgenic plants. In addition, we also made double RNAi constructs for two groups of genes given that these mutants had no visible phenotype probably due to gene redundancy. We have completed the analysis of nucleosome position mapping in six different rice cultivars and carried out comparison analysis among the six different rice cultivars. We completed RAN-seq and data analysis in the same tissue for these six rice cultivars used for nucleosome positioning analysis. The correlation between nucleosome positioning and gene expression has been investigated. Our results showed that nucleosome positioning follows the same pattern as other eukaryotic cells, for example, depleted in front of the TSS sites and enriched immediately following the TSS sites. Our research substantially advanced the understanding of role of SWI/SNF remodeling complex in gene expression and development in plants. Further understanding of the regulatory mechanism should have broad application in agriculture.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
1) Nallamilli, B.R.R., Zhang, J., Hana Mujahid, H., Brandon M. Malone, B. M., Susan M. Bridges, S. M., and Peng, Z. (2013) Polycomb Group Gene OsFIE2 Regulates Endosperm Development and Grain Filling in Rice (Oryza sativa) via a Mechanism Distinct from Arabidopsis. PLOS Genetics, 9, e1003322.
2) Nallamilli, B.R.R., Mariola Edelmann, Hana Mujahid,Zhaohua Peng (2014) Global Analysis of Lysine Acetylation Suggests the Involvement of Protein Acetylation in Diverse Biological Processes in Rice (Oryza sativa). 9(2):e89283.
3) Mujahid, H,, Tan, F,, Zhang, J., Nallamilli, B.R., Pendarvis, K., Peng, Z. (2013) Nuclear proteome response to cell wall removal in rice (Oryza sativa). Proteome Sci. 19;11(1):26.
4) Jian Zhang, Hana Mujahid, Yuxuan Hou, Babi R. Nallamilli, Zhaohua Peng (2013) Plant Long ncRNAs: A New Frontier for Gene Regulatory. Vol 4, PP. 1038-1045.
5) Malone, B.M., Tan, F., Bridges, S.M., Peng, Z. (2011) Comparison of four ChIP-Seq analytical algorithms using rice endosperm H3K27 trimethylation profiling data. PLoS One. 6(9):e25260.
6) La, H,, Ding, B., Mishra, G. P., Zhou, B., Yang, H., Bellizzi, R., Chen, S., Meyers, B. C., Peng, Z., Zhu, J. K., Wang, G. L. (2011) A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice. Proc Natl Acad Sci U S A. 108(37):15498-503.
7) Yuanda Lv1, 2, Zhikai Liang2, Min Ge1, Weicong Qi, Tifu Zhang1, Feng Lin1, Zhaohua Peng2,* and Han Zhao1,*Genome-wide identification and functional prediction of nitrogen-responsive intergenic and intronic lncRNAs in maize. BMC Genomics, In press. *co-corresponding author.
Oral Presentation
1) Zhaohua Peng (2013) Epigenetic Regulation of Endosperm Development in Rice (Oryza sativa), JianSu University, Zhenjiang city, JiangSu, China, Dec 24, 2013.
2) Zhaohua Peng (2013) Global protein acetylation in rice, Jiangsu Academy of Agricultural Sciences, JiangSu, China, Dec 26, 2013.
3) Zhaohua Peng (2013) Role of Histone Modifications in the Regulation of Endosperm Development in Rice (Oryza sativa), Instititute of Biotechnology, Jiangsu Academy of Agricultural Sciences, Dec 28, 2013.
4) Zhaohua Peng, Babi Ramesh Nallamilli, and Jian Zhang (2012) Genetic and Epigenetic Regulation of Seed Development and Grain Filling in Rice (Oryza sativa). Rice Technical Working Group 34rd Meeting, February 27-March 1, Hot Springs, Arkansas.
5) Babi Ramesh Nallamilli and Zhaohua Peng (2012) Genome Wide Identification of Protein Acetylation in Rice (Oryza sativa). Rice Technical Working Group 34rd Meeting, February 27-March 1, Hot Springs, Arkansas.
6) Zhaohua (2011) Regulation of Rice Endosperm Development and Grain filling role of Histone Modifications. July 15, Dale Bumpers National Rice Research Center, USDA, Stuttgart, AR.
Poster
1) Qing Miao, Babi Ramesh Nallamilli, Yuanda Lv, Han Zhao, Xing-Wang Deng, and Zhaohua Peng (2013) The Role of SWI/SNF Chromatin Remodeling Complexes in the Regulation of Grain Filling in Rice (Oryza sativa), 100. May 22-23, Washington DC.
2) Zhaohua Peng, Babi Ramesh Nallamilli, and Jian Zhang (2012) Genetic and Epigenetic Regulation of Seed Development and Grain Filling in Rice (Oryza sativa). Rice Technical Working Group 34rd Meeting, February 27-March 1, Hot Springs, Arkansas.
3) Babi Ramesh Nallamilli and Zhaohua Peng (2012) Genome Wide Identification of Protein Acetylation in Rice (Oryza sativa). Rice Technical Working Group 34rd Meeting, February 27-March 1, Hot Springs, Arkansas.
4) Babi Ramesh Reddy Nallamilli, Zhaohua Peng (2012) Polycomb group gene osfie2 plays an essential regulatory role in rice (Oryza sativa) endosperm development and grain filling. P15007. July 20-24, Austin, Texas.
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Progress 02/15/14 to 02/14/15
Outputs
Target Audience:Plant scientists, genetists, rice industry, rice breeders, Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One Ph D. student got trained. In addition, our research project was presented in molecular biology, molecular genetics, and senior seminar courses at Mississippi State University. Over one hundred graduate and undergraduate students learned more about chromatin remodeling and its role in rice seed development. How have the results been disseminated to communities of interest?We published related papers and presented our results to students in seminar classes and other related classes. What do you plan to do during the next reporting period to accomplish the goals?We plan to finish the project and got all the data published asap.
Impacts
What was accomplished under these goals?
We collected 34 putative mutants from rice T-DNA and transposon mutagenesis libraries worldwide for the chromatin remodeling genes. We carried out genotyping for all the putative mutants using PCR. We found that 18 of 34 putative mutants were true mutants and 16 of them were false mutants. Among 18 mutants, 14 had homozygous lines and 4 had only heterozygous lines. Phenotypes were observed in multiple generations in at least two of them. Due to gene redundancy, we did not see distinguished phenotype in most of the mutants. To solve the problem, we have constructed multiple RNAi double-knockout constructs. At least two constructs displayed severe phenotype in multiple plant tissues. We completed RAN-seq and data analysis for six RNA samples. We also completed the study of nucleosome positioning in the corresponding rice tissues. The correlation between nucleosome positioning and gene expression is under investigation. We have completed the analysis of nucleosome positioning in different rice tissues and different cultivars. We are performing comparison analyses among different rice tissues and rice cultivars.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Nallamilli, B.R.R., Mariola Edelmann, Hana Mujahid, Zhaohua Peng (2014) Global Analysis of Lysine Acetylation Suggests the Involvement of Protein Acetylation in Diverse Biological Processes in Rice (Oryza sativa).PLOS ONE, 9(2):e89283
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Progress 02/15/13 to 02/14/14
Outputs
Target Audience: plant sciencists, molecular biologiests, people who are interested in rice research and production, colleage students, postdoctoral fellows Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Four Ph.D. program graduate students are involved in the project. They are Qing Miao (MSU), Babi Ramesh Nallamilli (MSU), Xiaoxi Meng (MSU), and Bosheng Li (Yale). One undergraduate student Sean Duke is also involved in the project. How have the results been disseminated to communities of interest? we disseminated our results via publications, data deposit to public database, meeting posters, and oral presentations What do you plan to do during the next reporting period to accomplish the goals? We will continue data analysis and manusciption preparations. We will continue to generate and characterize the mutants in the chromatin remodeling pathways in rice. Finally, we will try to purify the chromatin remodeling complex again.
Impacts
What was accomplished under these goals?
Proposed Objectives: 1) To generate mutants for all the 11 putative genes encoding the subunits of SWI/SNF complex in rice and examine their role in the regulation of storage nutrient quality and quantity in rice. The content and properties of storage proteins, starch, and lipids in these mutants will be examined. Accomplishment: We collected 34 putative mutants from rice T-DNA and transposon mutagenesis libraries worldwide for the chromatin remodeling genes. We carried out genotyping for all the putative mutants using PCR. We found that 18 of 34 putative mutants were true mutants and 16 of them were false mutants. Among 18 mutants, 14 had homozygous lines and 4 had only heterozygous lines. Five of the 14 homozygous mutants had putative visible phenotype. The phenotypes were confirmed by multiple generation observations in at least two of them. Due to gene redundancy, we did not see distinguished phenotype in many mutants. Therefore, we are making double and triple mutants by crossing and RNAi approach. Proposed Objectives: 2) To study the role of the SWI/SNF complex in transcriptional regulation by profiling the transcriptome of selected mutants with an interesting nutrition phenotype. Accomplishment: We completed RAN-seq and data analysis for six RNA samples. We also completed the study of nucleosome positioning in the corresponding rice tissues. The correlation between nucleosome positioning and gene expression is under investigation. Proposed Objectives: 3) To map the binding sites of SWI/SNF complex in both young seedlings and developing seeds. Accomplishment: We are studying the nucleosome positioning in different rice tissues and different cultivars. We have completed mononucleosome generation, purification, DNA isolation, high throughput sequencing and data analysis. We are comparing nucleosome positioning among different rice tissues and rice cultivars. Once the SWI/SNF mutant characterization is completed, we will compare the nucleosome positions between wildtype and the mutants. Thus, the target of the SWI/SNF complex in vivo will be revealed. Proposed Objectives: 4) To purify and characterize SWI/SNF complex in young seedlings and developing seeds. Tandem affinity purification and other complementary methods will be used. Accomplishment: We tried to purify the SWI/SNF complex using tandem affinity tagged OsNSF5. Our results suggest that the tagged protein is highly unstable. We made another construct to tag another subunit of the SWI/SNF complex. Plant transformation is in progress. We hope that the newly tagged subunit is a stable core of the complex. Therefore, the complex can be purified using affinity columns.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
1) Nallamilli, B.R.R., Zhang, J., Hana Mujahid, H., Brandon M. Malone, B. M., Susan M. Bridges, S. M., and Peng, Z. (2013) Polycomb Group Gene OsFIE2 Regulates Endosperm Development and Grain Filling in Rice (Oryza sativa) via a Mechanism Distinct from Arabidopsis. PLOS Genetics, 9, e1003322.
2) Nallamilli, B.R.R., Mariola Edelmann, Hana Mujahid,Zhaohua Peng (2014) Global Analysis of Lysine Acetylation Suggests the Involvement of Protein Acetylation in Diverse Biological Processes in Rice (Oryza sativa). 9(2):e89283.
3) Mujahid, H,, Tan, F,, Zhang, J., Nallamilli, B.R., Pendarvis, K., Peng, Z. (2013) Nuclear proteome response to cell wall removal in rice (Oryza sativa). Proteome Sci. 19;11(1):26.
4) Jian Zhang, Hana Mujahid, Yuxuan Hou, Babi R. Nallamilli, Zhaohua Peng (2013) Plant Long ncRNAs: A New Frontier for Gene Regulatory. Vol 4, PP. 1038-1045.
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Progress 02/15/12 to 02/14/13
Outputs
OUTPUTS: The project aims to study the biological function of chromatin remodeling genes in rice with emphasis on grain development. 1) Study the mutants of chromatin remodeling genes Accomplishment:We collected 38 putative mutants from rice T-DNA and transposon mutagenesis libraries worldwide for the chromatin remodeling genes. We carried out genotyping for all the putative mutants using PCR. We found that 19 of 38 putative mutants were true mutants, 17 of them were false mutants, the other two remains to be further verified. Among the 38 mutants, we observed phenotype initially in 12 mutants. However, among the 12 mutants with phenotype, only 5 are true mutants.We are further characterizing these mutants. 2) Transcriptome Analysis Accomplishment: We completed RAN-seq analysis for six RNA samples. The RNA-Seq data analysis pipeline has been established. We are still characterizing some of the mutants. We will carry out transcriptome analysis in selected mutants after other background mutations have been removed. 3) Mapping the binding sites of the SWI/SNF complex Accomplishment: We have completed mononucleosome generation and purification studies. High throughput DNA sequencing of the nucleosome DNA has been completed. The data analysis pipeline has been established. We are organizing the data for a manuscript. 4) Proteomics study of the SWI/SNF complex We generated OsNSF5 tandem affinity tagged transgenic lines for SWI/SNF complex purification. We tried to purify the SWI/SNF complex using tandem affinity purification approach. Our results suggest that the SWI/SNF complex is highly unstable. While the OsSNF5 protein was abundant in the first purification step, the protein became barely detectable in the second purification step, suggesting a rapid degradation of the protein complex. We are optimizing the purification conditions to further purify the protein complex. PARTICIPANTS: Babi Ramesh Reddy Nallamilli Qing Miao, Zhaohua Peng, Xingwang Deng TARGET AUDIENCES: The PD taught about chromatin remodeling in his Epigenetic course. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Cromatin remodeling is a very important epigenetic regulatory mechanism of eukaryotic cells. However, the biological functions of chromatin remodeling genes are poorly explored in rice. Our study will generate the mutants of chromatin remodeling genes and establish the map of SWI/SNF complex binding sites genome wide. Our results will have critical reference value for understanding the epigenetic regulatory mechanism on gene expression.
Publications
- Babi Ramesh Reddy Nallamilli1, Jian Zhang1, Hana Mujahid1, Brandon M. Malone2,3, Susan M. Bridges2,3 and Zhaohua Peng1* (2013) Polycomb Group Gene OsFIE2 Plays an Essential Regulatory Role in Rice (Oryza sativa) Endosperm Development and Grain Filling. Plos Genetics. 9, e1003322.
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Progress 02/15/11 to 02/14/12
Outputs
OUTPUTS: We collected 34 putative mutants from rice T-DNA and transposon mutagenesis libraries worldwide for the chromatin remodeling genes. We carried out genotyping for all the putative mutants using PCR. We found that 18 of 34 putative mutants were true mutants and 16 of them were false mutants. Among 18 mutants, 14 had homozygous lines and 4 had only heterozygous lines. Five of the 14 homozygous mutants had putative phenotype in seed setting or seed filling. We are growing the putative mutants for further characterization including seed phenotype such as storage proteins, starch, and lipids. We are performing RAN-seq analysis for six RNA samples. We have completed library construction and next generation RNA sequencing. Data analysis just started. The purpose of the study is to correlate the nucleosome positioning with gene expression in rice. Once the RNA-Seq analysis pipeline has established and the mutant characteristics are re-verified, we will perform transcriptome studies for the selected mutants. We are studying the nucleosome positions in different rice tissues. We have completed mononucleosome generation and purification studies in five different rice tissues including young endosperm, filling stage endosperm, flower, leaf, and calli. High throughput DNA sequencing of the nucleosome DNA has completed in these five tissues. The data analysis pipeline has established with further adjustment expected. Completion of the study will generate position map in plants. Once the SWI/SNF mutants are verified, we will compare the nucleosome positions between wildtype and the mutants. Thus, the target of the SWI/SNF complex in vivo will be revealed. In addition, we have established ChIP-Seq data analysis pipeline (Malone, et al., 2011). The OsNSF5 tagged transgenic lines has also been generated. If the binding sites of the SWI/SNF can't be revealed by comparing the nucleosome position between wildtype and mutants, we will carry out ChIP-Seq experiments as proposed originally. We believe that compare the nucleosome position between mutants and wildtype can reveal function of the remodeling complex more directly because the false positively can be removed by subtraction. We tried to purify the SWI/SNF complex using tandem affinity purification approach. The OsNSF5 protein was tagged by generating transgenic lines and the transgenic plants were used for protein purification with a standard procedure. Our results suggest that the SWI/SNF complex is highly unstable. While the OsSNF5 protein was abundant in the first purification step, the protein became barely detectable in the second purification step, suggesting a rapid degradation of the protein complex. We are optimizing the purification conditions to further purify the protein complex. PARTICIPANTS: Project Director: Zhaohua Peng, Associate Professor Dept. of Biochemistry and Molecular Biology, Mississippi State University Co-PDs: Xing-Wang Deng, Professor Dept. of MCDB, Yale University Three Ph.D. program graduate students are involved in the project. Qing Miao, Mississippi State University Babi Ramesh Nallamilli, Mississippi State University Bosheng Li, Yale University One undergraduate student is also involved in the project. Sean Duke, Mississippi State University TARGET AUDIENCES: Plant scientists, epigenetists, molecular Biologists, rice breeders, rice farmers, and students are expected to be benefited from the data and knowledge generated in the research. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Mutants of the SWI/SNF complex genes have been identified. Putative phenotypes were observed. Characterization of the mutants are still in progress. Interesting mutants observed in this project is now used for rice breeding. Genome wide maps of nucleosome positioning in different tissues are expected to be delivered soon.
Publications
- 1) Malone, B.M., Tan, F., Bridges, S.M., Peng, Z. (2011) Comparison of four ChIP-Seq analytical algorithms using rice endosperm H3K27 trimethylation profiling data. PLoS One. 2011;6(9):e25260. Epub 2011 Sep 30.
- Peng, Z., Nallamilli, B.R., and Zhang, J. (2012) Genetic and Epigenetic Regulation of Seed Development and Grain Filling in Rice (Oryza sativa). Proceedings of the 34 Rice Technology Working Group Meeting. Feb 28, Hot Spring, Arkansas.
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