Elucidating the Gene Regulatory Networks that Specify Invasive Behavior

描述（申请人提供）：我的长期目标是在一流的研究机构中成为一名独立的科学家，研究在发育和癌症过程中调节细胞形态发生运动的基因调控网络（grn）。为此，拟议的研究促进了我在功能基因组学和系统生物学方法方面的培训，补充了我以前在细胞和发育生物学技术方面的研究。grn包括转录因子（tf）之间以及tf与其驱动特定细胞生物学过程的靶基因之间的物理和调控关系。尽管它们在调节细胞侵袭行为方面很重要，但TFs及其指定侵袭性的下游靶标的身份在很大程度上是未知的。细胞通过基底膜的侵袭在正常发育、免疫监视过程中是细胞扩散和器官形成的机制，在癌症转移过程中被错误调控。杜克大学Sherwood实验室建立了一个简单的体内模型，该模型独特地结合了强大的遗传，功能基因组学和单细胞视觉分析，可以分析秀丽隐杆线虫幼虫发育过程中锚定细胞（AC）通过BM侵入外阴上皮。我们目前对AC侵袭的理解包括鉴定出三种tf和少量假定的下游靶点，这些靶点既调节专门侵袭膜的建立，也调节AC在侵袭过程中清除BM的能力。在该奖项的指导阶段，拟进行的实验将开始通过BM表征grn潜在的细胞入侵：1)鉴定bZIP致癌基因TF的结合伴侣，fos-1a，以及线虫基因组中fos-1a结合位点的鉴定；2)鉴定其他调节AC侵袭的tf，并检测它们在体外是否具有调节癌细胞侵袭的保守功能。功能基因组技术和系统生物学方法的培训将通过高级课程、参加全国会议、与Marian Walhout博士的实验室（马萨诸塞大学医学院）合作以及杜克大学系统生物学小组和基因组科学与政策研究所（IGSP）主任Philip Benfey博士的共同指导来加速。人类癌症体外试验的培训将在Stephen Weiss博士（密歇根大学）的实验室进行。这些培训经验和在指导阶段获得的数据将为开展独立研究事业提供基础。在该奖项的未指导阶段提出的研究包括：1)表征包含在侵袭过程中控制AC基因表达的tf结合位点的基因组调控区域；2)确定在正常发育和人类癌症侵袭过程中重现侵袭程序所必需和充分的tf核心补体。

DESCRIPTION (provided by applicant): My long-term goal is to establish myself as an independent scientist at a top-tier research institution studying the gene regulatory networks (GRNs) that regulate cell morphogenetic movements during development and cancer. Towards this end, the proposed research advances my training in functional genomics and systems biology approaches that complement my previous research in cellular and developmental biology techniques. GRNs encompass both the physical and regulatory relationships amongst transcription factors (TFs) and between TFs and their target genes that drive specific cell biological processes. Despite their importance in regulating cell invasive behavior, the TFs and the identity of their downstream targets that specify invasiveness is largely unknown. Cell invasion through basement membrane (BM) serves as a mechanism underlying cell dispersal and organ formation during normal development, immune surveillance, and is mis-regulated during cancer metastasis. The Sherwood laboratory at Duke University has established a simple in vivo model that uniquely combines powerful genetic, functional genomic, and single cell visual analyses of anchor cell (AC) invasion through BM into the vulval epithelium during C. elegans larval development. Our current understanding of AC invasion includes the identification of three TFs and a handful of putative downstream targets that regulate both the establishment of a specialized invasive membrane and the ability of the AC to remove BM during invasion. The proposed experiments during the mentored phase of the award will begin to characterize the GRNs underlying cell invasion through BM by 1) identifying the binding partner of the bZIP oncogene TF, fos-1a, and the identity of FOS-1A binding sites within the C. elegans genome; and 2) identifying additional TFs that regulate AC invasion and testing whether they show conserved functions in regulating carcinoma cell invasion in vitro. Training in functional genomic techniques and systems biology approaches will be accelerated through advanced course work, attendance at national conferences, a collaboration with Dr. Marian Walhout's laboratory (University of Massachusetts Medical School), and co-mentorship by Dr. Philip Benfey, the director of Duke University's Systems Biology group and the Institute for Genome Sciences and Policy (IGSP). Training in human cancer in vitro assays will be carried out in the laboratory of Dr. Stephen Weiss (University of Michigan). These training experiences and the data acquired during the mentored phase will provide the basis to launch an independent research career. The research proposed in the unmentored phase of the award includes 1) characterizing the genomic regulatory regions that contain TF-binding sites that control gene expression in the AC during invasion and 2) determining the core complement of TFs that are both necessary and sufficient to recapitulate an invasion program in normal development and in human cancer invasion.