RUI: Functional Analysis of the C. elegans Winged-Helix Transcription Factor, LIN-31

LIN-31蛋白是翼螺旋转录因子家族的一员，在秀丽隐杆线虫发育过程中对外阴细胞命运的适当规范是必需的。该蛋白被认为在发育过程中起两个作用：(1)促进外阴细胞命运（当被信号转导蛋白MAP激酶磷酸化时）；(2)促进非外阴细胞命运（当与另一种转录因子LIN-1异二聚化时）。本项目旨在通过对这种多面蛋白的功能分析，进一步研究LIN-31在外阴发育过程中细胞命运规范中的作用。本项目的具体目标是：(1)翼螺旋转录因子LIN-31的功能分析（定点诱变实验将为LIN-31蛋白的不同区域分配功能），(2)LIN-31::LIN-1异二聚化元件的鉴定（共免疫沉淀实验将用于确定LIN-31蛋白的酸性区域是否需要与LIN-1蛋白（已知的异二聚化伙伴）进行异二聚化），(3)鉴定高亲和力的LIN-31结合序列（序列选择技术将用于鉴定高亲和力结合LIN-31 DNA结合域的寡核苷酸，然后这些序列将用于鉴定LIN-31转录因子的实际靶标）。这个项目的智力价值是通过提高我们对决定细胞命运的信号级联末端转录因子的认识，更好地理解基因调控如何控制发育。此外，通过确定该转录因子的潜在靶点，本提案中描述的实验也将阐明秀丽隐杆线虫外阴细胞命运途径中尚不清楚的部分。该项目的更广泛影响包括对教学、培训和学习的重大贡献。本科生将是项目的重要组成部分，私家侦探参与研究将提高她的教学水平，培养教学博士后将为教育和研究提供重要的培训，并加强SCU不断发展的“科学学者群体”。此外，迄今为止在P.I.进行研究的23名本科生中其中17名（74%）是女性，其中大多数已经攻读了研究生课程。这些数字表明，在招募和教育代表性不足的群体成员方面取得了成功。SCU也有大量的拉丁裔人口，将继续招收研究学生。

0315072MillerThe LIN-31 protein, a member of the winged-helix family of transcription factors, is required for the proper specification of vulval cell fates during C. elegans development. This protein is believed to play two roles during development: (1) to promote vulval cell fates (when phosphorylated by the signal transduction protein MAP kinase) and (2) to promote non-vulval cell fates (when heterodimerized with another transcription factor, LIN-1). This project aims to further investigate LIN-31's role in cell fate specification during vulval development by continuing a functional analysis of this multifaceted protein.The Specific Aims of this proposal are (1) Functional Analysis of the Winged Helix Transcription Factor LIN-31 (site-directed mutagenesis experiments will assign functions to different regions of the LIN-31 protein), (2) Identification of LIN- 31::LIN-1 Heterodimerization Elements (co-immunoprecipitation experiments will be used to determine if the acidic region of the LIN-31 protein is required for heterodimerization with the LIN-1 protein, a known heterodimerization partner), and (3) Identification of High-Affinity LIN-31 Binding Sequences (a sequential selection technique will be used to identify oligonucleotides that bind the LIN-31 DNA binding domain with high affinity--those sequences will then be used to identify actual targets of the LIN-31 transcription factor).The Intellectual Merit of this project is to provide a better understanding of how gene regulation controls development by advancing our knowledge of a transcription factor at the end of a signaling cascade that determines cell fates. In addition, by identifying potential targets of this transcription factor, the experiments described in this proposal will also illuminate a part of the C. elegans vulval cell fate pathway that is not well understood. The Broader Impacts of this project include significant contributions to teaching, training and learning. Undergraduate students will be an integral part of the project, involvement of the P.I. in research will enhance her teaching, and training of a Teaching Post-Doc will provide critical training in education and research, in addition to enhancing the evolving "community of science scholars" at SCU. Furthermore, of the 23 undergraduate students who have thus far conducted research in the P.I.'s laboratory, 17 (74%) were women, most of which have gone on to post-graduate programs. These numbers indicate established success in recruiting and educating members of underrepresented groups. SCU also has a significant Latino population, from which research students will continue to be recruited.