A Novel Post-transcriptional Regulatory Mechanism Mediated by Zhx2

智力优势：越来越清楚的是，基因表达的转录和转录后步骤是耦合的、相互依赖的，并且可能是共同调节的。通过与转录结合来调节信使核糖核酸的加工的许多证据都来自人工系统。虽然这些模型证明了通过与转录机制的相互作用来调节基因表达的转录后步骤的能力，但在体内发生这种情况的自然例子很少。最近，锌指和同源盒2(ZZX2)基因被克隆，并被证明在一定程度上抑制了甲胎蛋白(AFP)和其他基因在出生后肝脏的表达。已发表的研究表明，AFP启动子足以对异源报告基因进行zhx2调控。然而，初步结果表明，在存在或不存在zhx2的情况下，AFP基因的转录速率是相同的，即使AFP mRNA的积累受到抑制。此外，这种抑制是由于多个AFP内含子的剪接被抑制而发生的。由于ZX2的功能依赖于启动子，但作用于转录后水平，该系统提供了一个独特的机会来理解转录和转录后基因调控之间的生物学相关和新的机制。在初步研究的基础上，假设zhx2通过其靶基因的启动子作用，降低了新生RNA的剪接效率，从而降低了完全加工的mRNA水平。为了验证这一假设并研究机制细节，已经产生了表达标记为zhx2的转基因但内源性zhx2水平较低的小鼠。利用这些小鼠，该项目将通过染色质免疫沉淀(ChIP)分析来确定zhx2是AFP表达的直接或间接调节因子，以及它是否与目标启动子或整个基因相关联。将芯片和DNA测序相结合，将识别其他受ZX2调控的基因。通过比较存在或不存在zhx2的情况下的polII复合体，将进一步研究AFP RNA剪接抑制的机制。剪接抑制是否以共转录方式发生，将通过测量新生RNA的剪接来确定。最后，该项目将确定与zhx2相互作用的蛋白质，这将提供关于zhx2功能的基本信息。成年小鼠肝脏中含有FLAG-ZX2的蛋白质复合体的组成将通过质谱仪进行鉴定。该项目的长期目标是了解zhx2介导的AFP调节，因为它是一种新的调节机制，似乎将转录和转录后事件耦合在肝脏中。这一机制的细节将有助于更好地理解RNA生物发生步骤中的基因调控。广泛的影响：研究生、本科生，以及潜在的高中生将参与这项研究，这是基于该实验室在学生培训方面的强大历史。研究成果将通过国家会议报告和出版物与更广泛的科学界分享。从科学上讲，这项研究有可能发现对鲜为人知的基因调控机制的新见解。

Intellectual Merit: It has become increasingly clear that transcriptional and post-transcriptional steps of gene expression are coupled, interdependent and potentially co-regulated. Much of the evidence for regulating mRNA processing through coupling with transcription is derived from artificial systems. While these models demonstrate the capacity for regulating post-transcriptional steps of gene expression through interactions with transcriptional machinery, there are few natural examples of where this occurs in vivo. Recently, the Zinc fingers and homeoboxes 2 (Zhx2) gene was cloned and shown to be responsible, in part, for repressing the expression of alpha-fetoprotein (AFP) and other genes in the liver after birth. Published studies have shown that the AFP promoter is sufficient to confer Zhx2 regulation on a heterologous reporter gene. However, preliminary results have shown that the transcription rate across the AFP gene is the same in the presence or absence of Zhx2, even though AFP mRNA accumulation is repressed. Moreover, this repression occurs because splicing of multiple AFP introns is inhibited. Because Zhx2 functions in a promoter-dependent manner but acts at a post-transcriptional level, this system provides a unique opportunity to understand a biologically relevant and novel mechanism that couples transcriptional to post-transcriptional gene regulation. Based on preliminary studies, it is hypothesized that Zhx2 acts through the promoter of its target genes to decrease the splicing efficiency of the nascent RNA and thus reduce fully processed mRNA levels. To test this hypothesis and investigate mechanistic details, mice that express a FLAG-tagged Zhx2 transgene, but low levels of endogenous Zhx2, have been generated. Using these mice, this project will determine whether Zhx2 is a direct or indirect regulator of AFP expression and whether it associates with target promoters or along the whole gene by using chromatin immunoprecipitation (ChIP) assays. Combining ChIP with DNA sequencing will identify other Zhx2-regulated genes. The mechanism of AFP RNA splicing repression will be investigated by comparing the pol II complex in the presence or absence of Zhx2 by ChIP. Whether splicing repression occurs co-transcriptionally will be determined by measuring splicing of nascent RNA. Finally, this project will identify proteins that interact with Zhx2, which will provide essential information about Zhx2 function. The components of FLAG-Zhx2-containing protein complexes from adult mouse liver will be identified by mass spectrophotometry. The long-term goal of the project is to understand Zhx2-mediated regulation of AFP because it is a novel regulatory mechanism that appears to couple transcription to post-transcriptional events in the liver. Details of this mechanism will lead to a better understanding of gene regulation at the step of RNA biogenesis.Broader impacts: Graduate, undergraduate, and, potentially, high school students will be involved in the research, building on the laboratory's strong history of student training. Results of the research will be shared with the broader scientific community through national meeting presentations and publications. Scientifically, the research has the potential to uncover novel insights into a poorly understood mechanism of gene regulation.