Yanhai Yin - US grants

The anterior pituitary gland develops from a midline structure called Rathke~s pouch into five cell types, corticotropes, gonadotropes, thryrotropes, somatotropes and lactotropes. The gland integrates complex feedback mechanisms , receiving information from the brain via the hypothalamus, signaling to peripheral endocrine organs, and thereby regulating such vital processes as metabolism, growth, reproduction and behavior. The mechanisms of many diseases occurring in these processes can be revealed by characterizing factors that control pituitary development. The long-term objectives of the proposal is to identify signal molecules and corresponding transcriptional factors that control the pituitary organ commitment and cell lineage specification. The specific aims of the proposal are 1) to characterize signal pathways that control pituitary commitment and cell lineage determination by transgenic studies: experiments are proposed to disrupt BMP-4, Wnt-5A and FGF8 signaling pathways implicated in pituitary development by transgenic expression of dominant negative forms of BMP4 and FGF8 receptors and wildtype GSK3 that impairs Wnt signaling 2)to characterize a novel forkhead/HNF-3 factor implicated in mediating BMP-4 signaling in pituitary development by targeted gene disruption through homologous recombination; 3) to characterize a novel bHLH factor implicated in cell type specification in pituitary: a novel bHLH transcriptional factor that expresses between e10-e16 during pituitary development have been identified and will be characterized genetically by creating knock-out mice through homologous recombination.

ABSTRACT NOT AVAILABLE

Yanhai Yin
0546503
CAREER: Novel Signaling Components For Plant Steroid Regulated Gene Expression
In Arabidopsis

Plant steroid hormones called brassinosteroids (BRs) control many diverse processes such as cell growth, senescence and stress responses. Application of BRs has been shown to increase crop yield and improve plant resistance to drought, high temperature and various diseases. Although significant progress has been made in identifying BR receptors and several other signaling components, little is known about how plant steroids regulate gene expression, thereby controlling different biological responses. Modern genetic and genomic approaches will be used to address this question. A BR-regulated transcription factor BES1 mediates many of the hormone responses and provides a powerful tool to study BR-regulated gene expression. To reveal the gene regulatory circuits, the functions of several BES1-induced transcription factors will be determined by over-expression and knock-out technologies. To identify other signaling components involved in BR-regulated gene expression, mutant plants with altered responses to plant steroids will be isolated, the underlying genes will be identified and characterized. The results from these studies will reveal the complex network through which BRs control growth and responses to both biotic and abiotic stresses. Broader impacts: Since BRs control many different biological processes, it is highly desirable to be able to manipulate individual responses. For example, it would be beneficial to promote BR-induced growth and stress responses and to reduce BR-induced senescence. The acquired knowledge from the proposed research can be used to achieve these goals. Several BR mutants will be introduced into an undergraduate Plant Physiology laboratory course to illustrate how steroids control plant growth. In addition, students from underrepresented groups will be recruited to work on the project through the Program for Woman in Science and Engineering (PWSE) at Iowa State University. The integrated research and teaching program will therefore provide ample training opportunities for high school, college, graduate and postgraduate students.

Plant steroid hormones, Brassinosteroids (BRs), play important roles in plant growth, development and response to environmental stresses. BRs act through several signaling components to regulate BES1/BZR1 and other families of transcription factors (TFs), which in turn control the expression of about 4000 genes required for various BR responses. How BR-regulated TFs control the expression of the large number of target genes and various BR responses is not well defined and will be the main focus of the proposal. The functions and mechanisms of several BR-regulated transcription factors (proteins responsible for activating or inactivating genes) will be studied by reverse genetic and genomic approaches and their regulation by BR signaling will be investigated by biochemical experiments. The results will reveal how each of the BR-regulated TFs controls a subset of BR target genes and a portion of BR responses. The studies will also provide important insight into how BR signal is integrated into the complex transcriptional network to control various BR-regulated processes. The knowledge obtained can be used to design strategies to improve the crop yield and tolerance to environmental stresses. Both mutants and transgenic lines generated from the project will be made available through the Arabidopsis Information Resource. In addition, the large-scale gene expression data will be deposited into NASC Arrays or GEO Datasets for wide dissemination. The proposed research will generate new knowledge that is essential for agriculture and bioenergy, especially under the constant environmental changes. In addition, a graduate student, research assistant and high school teacher will be trained to carry out the proposed experiments as a coordinated and cohesive team. The high school biology teacher will also be trained to bring some of the BR mutants to high school biology classes. The proposal, therefore, has a great potential in training future scientists and educators.

The increasing demand for food, feed and biofuel makes it very important to increase crop production, especially under adverse climate conditions. Plant steroid hormones, Brassinosteroids (BRs), have the potential to promote crop yield and improve plant performance against bacterial and fungal diseases and stresses such as drought and high temperature. Understanding BR functions in plants is essential to fully harvest its beneficial effects. Although BR signaling pathway has been well established, how BR regulates thousands of genes for various hormonal responses is not well understood. In this project, the investigators will use genetic, genomic, and computational approaches to establish gene regulatory networks through which BRs control the expression of the large number of genes for plant growth and stress tolerance. While the genetic and genomic studies will define the gene functions, the computational approach is required to build models for how hundreds to thousands of genes coordinate to carry out specific functions. The combination of functional genomic studies and computational modeling in establishing and validating transcriptional networks in hormone signaling is novel and is anticipated to provide unprecedented new insights into BR signaling network. The knowledge can be applied to agriculture to increase crop production. Moreover, the computational and bioinformatic tools developed and validated in the context of this project will be applicable to other systems biology studies. The research will also provide outstanding training opportunities at the interface of functional genomics and computational modeling for graduate and undergraduate students. High school science teachers will be recruited to participate in the research and to integrate such research into high school science education. The BR mutants will be introduced to an undergraduate laboratory course, in which a large number of undergraduate students each year will design and perform experiments to learn how plant steroid hormones control plant growth.

Project Summary The goal of this project is to determine how growth, development and stress responses are coordinated in Arabidopsis, a model plant with extensive genetic, genomic and proteomic resources. This will be accomplished through detailed mechanistic studies that will provide insights into fundamental biological processes, steroid hormone signaling and autophagy, that are conserved across eukaryotes. Brassinosteroids (BRs) are plant steroid hormones that promote growth. Autophagy occurs in both plants and animals to degrade organelles and proteins, especially under stress conditions. Our preliminary work has established several interaction points between BR and autophagy pathways. First, BES1, a transcription factor mediating BR responses, is degraded by selective autophagy, mediated by the ubiquitin receptor DSK2. Furthermore, phosphorylation of DSK2 by BIN2, a negative regulator in the BR signaling pathway, increases the interaction between DSK2 and ATG8, resulting in BES1 degradation. Second, TOR, a negative regulator of autophagy, is required for BR-mediated growth, and BRs inhibit autophagy likely via BIN2 interaction with TOR. We hypothesize that BR and autophagy pathways crosstalk through multiple mechanisms to coordinate plant growth and stress responses: (a) upon phosphorylation by BIN2, DSK2 acts as a phospho-regulated autophagy receptor for BES1, and BES1 ubiquitination therefore leads to its degradation by selective autophagy. This in turn slows down plant growth under stress conditions; (b) BRs regulate TOR to promote growth and inhibit autophagy through BIN2 phosphorylation of TOR. To test and expand on these hypotheses we propose the following two Specific Aims: (1) To establish the functions of selective autophagy receptor DSK2 and E3 ubiquitin ligases BAF1 and BAR1 in mediating BES1 degradation through autophagy; (2) To determine the mechanism of BR regulation of TOR via BIN2, and the effect of this regulation on growth and autophagy under normal and stress conditions. These studies will leverage the genetic and genomic resources in Arabidopsis and use cutting-edge proteomics to study ubiquitination and phosphorylation at the individual protein and proteome-wide levels. These innovative approaches have the potential not only to reveal specific mechanisms of crosstalk between steroid signaling and protein degradation pathways, but also provide transformative concepts and information on the integration of growth and stress responses across eukaryotes. For example, autophagy is involved in many human diseases including neurodegenerative diseases (e.g. Amyotrophic Lateral Sclerosis, Parkinson's and Huntington's) and cancer. In addition, the degradation of BES1 by autophagy is reminiscent of that of ?-catenin in WNT signaling and HIF2? in hypoxia responses, which play essential roles in growth, development, stress responses and disease in animals and humans. The proposed studies can therefore provide important insight into processes related to human health.

Plant steroid hormones called Brassinosteroids (BRs) signal to regulate plant growth, development and response environmental stresses. However, our understanding of how this steroid hormone controls thousands of genes to regulate these processes is incomplete. The project will generate and use multiple large-scale datasets to model how genes are controlled to govern BR-regulated growth and drought responses. BR pathways are well conserved among Arabidopsis and crop plants such as maize, rice and other crops, so the knowledge obtained from the proposed studies can be used to design strategies to optimize plant growth and crop production under drought conditions. Moreover, the project provides excellent training opportunities for graduate students and postdoctoral associates in modern plant biology. The investigators will also achieve societally relevant outcomes by providing training opportunities to underrepresented undergraduates through the George Washington Carver Internship program as well as engaging high school and community college teachers via summer internships. Through this partnership with teachers a learning module on BR-regulation of plant growth will be developed for high-school and community college students. Furthermore, unpublished data from the proposed studies will be incorporated into an upper level undergraduate course on functional genomics, systems, and network biology, freeing students to generate their own hypotheses during a group project rather than simply reproducing published work. Finally, the investigators will provide resources to the research community by organizing a workshop on plant phenomics, proteomics and computational modeling approaches and by making the data generated publicly accessible through publications and the Principal Investigator's laboratory website.

Molecular genetic studies in Arabidopsis have greatly advanced our understanding of the BR signaling pathway. BRs signal to regulate BES1/BZR1 family transcription factors (TFs), hundreds of BR-Related Transcription Factors (BR-TFs) and thousands of target genes. Although numerous BR-TFs have been identified, the transcriptional complexes that allow BES1/BZR1 and these BR-TFs to regulate the large number of BR-responsive genes have not been characterized. In addition, modeling of BR networks has proven to be a powerful approach to understand how BES1 directs a network controlling thousands of BR responsive genes. However, previously constructed BR networks have only considered transcription, overlooking important regulation at the post-transcriptional and post-translational level. The project will use an integrated genetics, genomics, and proteomics approach to establish and experimentally test a comprehensive Arabidopsis Gene Regulatory Network (GRN) governing BR-regulated growth and drought responses. By combining cutting-edge proteomics with advanced predictive modeling the project will generate a comprehensive view of BR-mediated transcriptional regulation and allow for the identification of novel factors involved in BR responses. First, investigators will uncover the components and functions of BR transcription factor complexes by examining the role of two novel BR-TFs. These two BR-TFs interact with BES1, a master regulator of the BR pathway, as well as a large number of other BR-TFs, likely forming large transcriptional complexes to control the expression of BR target genes. Second, investigators will generate BR transcriptome, proteome, and phosphoproteome datasets that are a prerequisite for a novel approach pioneered by the Co-Principal Investigator to construct GRNs based on these combined omics data. These combined networks have increased predictive ability compared to networks based only on transcriptional data and will provide a vital resource, allowing for a more complete understanding of the transcriptional program through which BRs control plant growth and stress responses.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.